Validation o f FHH-GhsrmlMcwi GHSR KO Rat as a

Model to Study Ghrelin Biology.

by

Valerie Rachelle Charbonneau

A thesis submitted to the Faculty of Graduate and Postdoctoral Affairs in partial fulfillment o f the requirements for the degree of

Master of Science

in

Neuroscience

Carleton University Ottawa, Ontario

©2012, Valerie Rachelle Charbonneau

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Abstract

Ghrelin, an orexigenic hormone peptide, is the endogenous ligand for the growth

hormone secretagogue receptor (GHSR). Previous research has established ghrelin as a

modulator in the regulation of appetitive behaviors and energy balance, and this research has

included the use of genetically altered mice with mutations to either the ghrelin or the GHSR

gene. Recently a rat model with a mutation to the GHSR gene has become available and the

present project attempted to validate this GHSR KO rat model. To this end, GHRS KO rats and

their wildtype (WT) littermates (n= 8/group) originally obtained from Transposagen Bio

(Lexington, KY) were single housed and monitored for food intake and body weight for a period

of 4 months. During the first 3 weeks, animals had access to standard chow, whereas the

remaining 3 months; animals were exposed to a High fat diet (60% calories from fat). Following

several experiments, our results show that, when correcting for body weight, GHSR KO rats ate

less and weighed less than WT littermates. Interestingly within the periphery, KO rats exhibited

lower triglyceride content relative to WT controls. With regards to the central nervous system,

our study was able to replicate previous findings reporting an increase in AgRP (an appetitive

regulating protein) mRNA expression. Collectively, our data reinforces the hypothesis that the

ghrelin receptor is indeed a potential target to prevent diet-induced obesity, and confirms the

GHSR KO rat is a valid model for the study of ghrelin and growth hormone secretagogue

receptor biology.

Acknowledgements

This project would not have been possible without the support of many people. First and

foremost, I offer my sincere gratitude to my MSc. Thesis supervisor, Dr. Alfonso Abizaid for his

continuous support, patience, motivation, enthusiasm, and immense knowledge. I am extremely

lucky to have a supervisor who demonstrated he cared about my work by responding to my

questions promptly, reading my numerous revisions and providing constructive criticism and

excellent advice during the preparation of this thesis. Without him, this would not have been

possible.

I am also grateful to other members of my thesis committee: Dr. Hymie Anisman and Dr.

Matthew Holahan who offered guidance and support throughout different stages o f my research.

I also wish to thank my fellow labmates: Harry MacKay for teaching me lab techniques

and great insight; Zack Patterson, Rim Khazall, Martin Wellman, Samantha King, Veronique St-

Onges and Trevor Rodrigues for sharing valuable advice and encouragement throughout this

process.

The financial support from Natural Sciences and Engineering Council o f Canada

(NSERC) for this thesis is greatly acknowledged. The Department o f Neuroscience at Carleton

University provided the equipment needed to produce and complete my thesis.

Last but not least, I owe my loving thanks to my family: my parents Louise and Claude

Charbonneau for their loving support and constant belief in me; my older brother Claude Jr.

Charbonneau for his knowledgeable guidance, and to my boyfriend Darren Michelutti for his

relentless support, understanding and encouragement.

Disclosure summary: The authors have nothing to disclose.

List of abbreviations

ACTH: Adrenocorticotropic hormone

AG: acylated ghrelin

AgRP: Agouti related peptide

Alpha-MSH: alpha menalocyte-stimulating hormone

ANOVA: Analysis of Variance

AP: area postrema

ARC: arcuate nucleus

AUC: Area under the curve

BDNF: brain-derived neurotrophic factor

CA: catecholamine

CART: cocaine-and amphetamine-regulated transcript

CNS: Central nervous system

DA: Dopamine

DIO: Diet-induced obesity

DMH: Dorsomedial nucleus of the hypothalamus

DMNV: dorsal motor nucleus of the vagus

Eliza- Enzyme-linked immunosorbent assay

ENU: ethyl-N-nitrosurea

FHHm'Mc*‘: fawn hood hypertensive mutation 1 Medical college of Wisconsin

FHL: fawn hooded low blood pressure

GH: growth hormone

GHRH: growth hormone releasing hormone

Ghrl: ghrelin

GHRL KO: Ghrelin knockout

GHSR KO: Growth hormone secretagogue receptor knockout

GOAT: ghrelin O acyltransferase

ICV: intracerebroventricular

IP: intraperitoneal

LH: lateral hypothalamic area

MC3/4R: melanocortin receptors 3 and 4

mRNA: messenger ribonucleic acid

NA: noradrenaline

Nac: Nucleus Accumbens

NPY: Neuropeptide

NTS: nucleus tractus solitarius

POMC: pro-opiomelanocortin

PVN: paraventricular nucleus

Qrt-PCR: quantitative real-time polymerase chain reaction

SN: substantia nigra

SYBR green:

TH: tyrosine hydroxylase

UAG: unacylated ghrelin

VMH: ventromedial nucleus of the hypothalamus

VTA: ventral tegmental area

WT: Wild type

iv

Table o f Contents

Abstract...........................................................................................................................................i

Acknowledgements.....................................................................................................................ii

List of Abbreviations................................................................................................................ iii

Table of Contents........................................................................................................................v

List of Illustrations........................................................................ vii

List of Appendices................................................................................................................... viii

1 Introduction........................................................................................................................... 1

1.1 Obesity Epidem ic............................................................................................................................1

1.2 Energy H om eostasis....................................................................................................................... 2

1.3 Ghrelin/GHSR/CNS system ......................................................................................................... 6

1.4 Previous Ghrl/GHSR KO mice find ings..................................................................................16

1.5 Hypothesis: Validating the GHSR KO Rat DIO M odel....................................................... 24

2 Methods.................................................................................................................................25

2.1 Fawn hooded rat background strain...........................................................................................25

2.2 Animal C a re .................................................................................................................................. 29

2.3 Generation o f GHSR KO rat .....................................................................................................29

2.4 Experimental D esign ....................................................................................................................30

3 Results....................................................................................................................................35

3.1 Experiment 1................................................................................................................................. 35

3.2 Experiment 2 ................................................................................................................................. 37

3.2 Experiment 2.1..............................................................................................................................40

3.3 Experiment 3................................................................................................................................. 41

3.3 Experiment 3.1..............................................................................................................................45

3.4 Experiment 4 ................................................................................................................................. 47

v

3.5 Experiment 5.................................................................................................................................. 48

4 Discussion................................................................................................................................52

4.1 Experiment 1.................................................................................................................................. 52

4.2 Experiment 2.................................................................................................................................. 53

4.2 Experiment 2.1............................................................................................................................... 53

4.3 Experiment 3...................................................................................................................................54

4.3 Experiment 3.1............................................................................................................................... 55

4.4 Experiment 4 .................................................................................................................................. 55

4.5 Experiment 5...................................................................................................................................56

4.6 Compensatory m echanism s.........................................................................................................59

4.7 Strengths......................................................................................................................................... 61

4.8 Limitations......................................................................................................................................61

4.9 Validation o f GHSR KO rat DIO m odel..................................................................................62

5 Conclusions............................................................................................................................. 52

Bibliography or References.................................................................................................... 65

vi

List of Illustrations

Figure 1. Experimental Design............................................................................................. 30

Figure 2a. Standard chow food intake measure in development period......................... 36

Figure 2b-c. Body weight in development period......................................................... 36-37

Figure 3. Standard chow food intake measure in adulthood........................................... 38

Figure 4a. Average body weight following standard chow diet in adulthood.............. 39

Figure 4b. Daily body weight gain on standard chow diet in adulthood.......................39

Figure 5. Overnight Fast..................................................................................................... .41

Figure 6. Food intake following 3 months of HFD in adulthood.................................. 42

Figure 7. Total daily average HFD intake normalized to body weight......................... 42

Figure 8. Body weight gain following 3 months of HFD................................................43

Figure 9. Average daily body weight gain on HFD........................................................ .43

Figure 10. Fat mass distribution following HFD............................................................44

Figure 11. Average total fat (g) mass following HFD.....................................................45

Figure 12. AUC measures following GTT during HFD................................................46

Figure 13. AUC curve.........................................................................................................46

Figure 14. Hypothalamic neuropeptide AgRP mRNA expression in ARC................ 47

Figure 15. Hypothalamic neuropeptide POMC mRNA expression in ARC............... .48

Figure 16. Fasting active plasma ghrelin content............................................................ .49

Figure 17. Fasting active plasma glucose content............................................................49

Figure 18. Fasting active plasma insulin content.............................................................50

Figure 19. Fasting active plasma leptin content............................................................. 51

Figure 20. Fasting active plasma triglyceride content.................................................. 51

List of AppendicesAppendices..................................................................................................................................80

Appendix A Previous DIO transgenic mice m odels............................................................................80

Appendix B Standard chow m acronutrients......................................................................................... 81

Appendix C High-Fat Diet macronutrients............................................................................................82

Appendix D qRT-PCR Prim ers...............................................................................................................83

viii

1 Introduction

1.1 Obesity Epidemic

Obesity and associated disorders including Type II diabetes and cardiovascular disease

represent one the biggest challenges to human health (Wang et al., 2006; Bloom et al., 2008;

Lobstein et al., 2004). In addition to posing a worldwide burden upon health care systems

(Bloom et al., 2008), the prevalence of obesity in children is also quickly escalating which

forecasts an even greater medical threat in the decades to come (Troiano & Flegal, 1999). As a

result of this epidemic, a peak of interest in obesity research has emerged in recent decades and

these efforts have led to the discovery of a number of hormones regulating appetite, and have

generated a certain optimism that drugs for the treatment o f obesity will soon follow. However,

enthusiasm has been premature and the exact molecular mechanism by which obesity develops

remains unclear. Current pharmacological treatments for obesity are either lacking in efficacy of

modulating relevant neuropeptides within pathways that control body weight or are burdened

with adverse side effects (Shellekens, 2010). Thus, novel strategies and research targets are

required for a better understanding of the intricate molecular pathways controlling energy

homeostasis (Shellekens et al., 2010).

One particular prospect is the growth hormone secretagogue receptor (GHSR), the only

known receptor for the orexigenic hormone ghrelin. A number of studies have identified this

receptor as a potential target for the pharmacological treatment o f obesity, and a number of

experimental models are currently being used to explore the biology of this receptor. Amongst

these models, there are a number of genetically altered mice models that have been used during

the past decade, and these have yielded useful information. These models, however, are difficult

1

to test at the behavioral level. Recently, genetically engineered rats have become available for

research, including a rat model with a targeted mutation to the GHSR. This rat model remains to

be fully characterized to demonstrate that it is a valid model of the GHSR mutation. Therefore,

the purpose of this project is to demonstrate that this rat model is indeed a valid model for the

study of GHSR and ghrelin biology.

1.2 Energy Homeostasis

Homeostasis is a fundamental process by which a system regulates its internal

environment to maintain a stable, constant condition of properties (Bernard; Cannon, 1926).

Homeostatic regulation allows an organism to effectively function in a broad range of

environmental conditions. Evidence of homeostatic mechanisms within the context o f regulating

energy balance and appetitive behaviors can be found throughout all cells and system in living

organisms. Feeding provides the body’s macronutrients (carbohydrates, lipids and proteins) and

most micronutrients (minerals and vitamins) that are critical for survival. Therefore feeding

behavior is one of the only behaviors we cannot eliminate if we begin to abuse it. For this

process to occur, the amount of energy consumed must match precisely the amount o f energy

expended to respect this law of energy balance. In order to manage and operate the entire

organism’s energy balance, there are molecular signals that are responsible to modulate food

intake while controlling short and long-term energy needs in storage (Woods et al., 1998; Saper

et al., 2002).

In vertebrates, a key center regulating energy homeostasis is the hypothalamus. The

hypothalamus is a relatively small central region composed of several nuclei and located at the

base of the brain where it can access signals from the periphery that send information about

water balance, nutrients, minerals, hormones, body temperature, and light/dark cycles.

2

In the case o f energy regulation, several nuclei within the hypothalamus are sensitive to

metabolic hormones and metabolic fuels like glucose and free fatty acids, and respond to

changes in these signals by altering metabolic rate and producing behaviors aimed at obtaining or

avoiding food to achieve homeostasis. In general, when nutrients are low or energy demands are

higher than usual, organisms are said to be in a negative energy state (i.e. when the cost o f life is

higher than the energy available), whereas when there is a surplus o f energy stores, the

organisms is said to be in a positive energy state. This is an important aspect o f energy

homeostasis whereby the body will store its fuel in the form of adipose tissue for future on-

demand needs (Woods et al., 1998). The hypothalamus is under constant stimulation

responding to either of these states to achieve a balance. It acts as a thermostat and regulates our

energy homeostasis integrating and interpreting peripheral tissue messages in order to output and

delegate the appropriate functions to other areas of the brain. As a result, the body is assigned to

act accordingly (i.e., manifest food-seeking behaviors when below homeostasis while lowering

energy expenditure for energy conserving purposes until homeostasis is recovered). This

concept requires a certain degree of plasticity to allow for constant dynamic changes from

stimuli and peripheral signals such as ghrelin and leptin continuously counter-regulating energy

balance (Abizaid, 2008). This flexibility develops responses to certain stimuli such that, hedonic

or palatable foods and cues are learned and remembered in the future. Proteins are commonly

found to rearrange neuronal connections in order for more efficient responses to future stimuli

(Abizaid et al., 2008). .

The critical role of the hypothalamus was determined by lesion studies. Of these, classic

studies included the destruction of the ventromedial nucleus o f the hypothalamus (VMH) using

electrolytic type of lesions and subsequently producing animals that became obese and diabetic

3

(Hetherington and Ranson, 1940). In other lesion studies, obesity and hyperphagia and insulin

resistance were also observed after destruction of other nuclei near the midline o f the brain

including the paraventricular nucleus o f the hypothalamus (PVN) and the dorsomedial nucleus of

the hypothalamus (DMH) (Brobeck, 1946). In contrast, lesions placed in the lateral

hypothalamus produced extreme anorexia (Powley and Keesey, 1970). In studies where they

stimulated these regions rather than ablating them, Valenstein et al., (1968) discovered a

decrease in food consumption when the VMH was stimulated whereas the opposite occurred

while stimulating the LH. This suggests that the medio-basal hypothalamus is important to

generate satiety and for metabolic regulation, whereas the lateral hypothalamus was important

for more orexigenic hunger mechanisms.

Arcuate Nucleus.

While the VMH, PVN, DMH and lateral hypothalamus are important for the regulation

of food intake and energy balance, the hypothalamic arcuate nucleus (ARC) has emerged as a

critical first order site where peripheral signals are integrated, and processed and projected to

change energy regulation.

First, the ARC resides outside o f the blood brain barrier, and as such is in direct contact with

circulating peripheral signals like hormones and nutrients. It also contains receptors for almost

every known metabolic hormone. There are several circuits within the hypothalamus that

contribute to its role in regulating appetite. One such circuit is found in the melanocortin system

(Flier et al., 2004). The ARC contains two sub-population of neurons that are critical for the

regulation of energy balance seeing as they are counter-regulatory (Flier et al., 2004). The first

group coexpresses neuropeptide Y (NPY) and a second orexigenic peptide, agouti-related

peptide (AgRP). Together, these neurons project to the PVN where NPY and AgRP have strong

4

effects on food intake and body temperature (Abizaid et al., 2008). They also stimulate the LH,

while sending inhibitory inputs to the VMH (Elmquist, 2001). The second sub neuronal group

coexpresses pro-opiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcript

(CART), both of which are anorexic peptides (Elias et al., 2001). In fact, POMC is a precursor

for several peptides, one of which is the a-menalocyte-stimulating hormone (a-MSH); a

neuropeptide that binds to melanocortin receptors 3 and 4 (MC3/4) in the hypothalamus to

reduce food intake and energy expenditure (Boston et al., 2007; Cone et al., 2001). Animals with

mutations to the POMC gene or to the MC3/4 receptors are leptin insensitive and morbidly obese

(Huzsar et al, 1997). Like NPY/AgRP neurons, POMC neurons also project to PVN and a-MSH

competes with AGRP for binding sites at MC3/4 receptors. Conversely to AgRP and NPY,

POMC cells have stimulatory inputs to the VMH and inhibitory inputs to the LH (Flier et al.,

2004).

Peripheral hormones regulating energy balance act on the melanocortin system to

modulate food intake and metabolism. For example, ghrelin acts on the ARC to stimulate the

activity o f NPY/ AgRP neurons, whereas leptin increases the activity of POMC and CART

neurons and the release o f their peptide products (Morton et al., 2001; Murphy et al., 2006).

Therefore, NPY/AgRP neurons activated by ghrelin stimulate feeding and decrease metabolic

rate and satiety, while POMC/CART neurons, activated by leptin, stimulate satiety and increase

metabolic rate while inhibiting feeding by acting on the NPY/AgRP cell group. Both

NPY/AGRP and POMC neurons project to second-order neurons expressing the melanocortin 4

receptor (MC4R). Interestingly, AGRP is a natural antagonist to the MC4R whereas a-

melanocyte stimulating hormone (a-MSH) (as previously mentioned to be the peptide derivative

o f the POMC protein) is a natural agonist to this receptor. Other than responding to leptin and

5

ghrelin signals, these two opposing cell groups communicate directly. Moreover, AgRP happens

to be a direct endogenous antagonist to the MC3/4 receptor (Oilman, et al., 1997). Because

targeted genetic disruption of MC4R in mice leads to increased food intake and increased lean

mass and linear growth it is thought that AgRP and a-MSH act against each other to maintain

homeostasis (Huszar et al., 1997). Hence, this appetite regulating system is very important for

constant regulation of energy balance.

1.3 Ghrelin/Arcuate Nucleus/ GHSR expression

Ghrelin and the regulation o f food intake and energy balance.

In humans and rodents, ghrelin is a 28 amino acid peptide and is the only peripheral

peptide known to increase appetite (Kojima et al., 1999; Delhanty & van der Lely 2011).

Ghrelin’s highest expression is found within the stomach, where it is syntehsized and secreted

from the X/A cells in the oxyntic gastric mucosa of the fundus (Delhanty et al., 2011). In

contrast to other gut peptides increasing after food ingestion, ghrelin peaks before food ingestion

as part of a mechanism that responds to fasting conditions. In addition, ghrelin secretion is

pulsatile and is released in larger quantities before meals suggesting that ghrelin is important for

meal initiation (Cummings et al., 2001; Kojima & Kanagawa 2005). Subsequently, ghrelin levels

fall as animals begin to eat, a physiological response that may contribute to satiety (Toshinai

et al. 2001). Ghrelin is composed of two forms. The most predominant form of ghrelin

circulating in the blood is unacylated (UAG), representing up to 90%, while acylated ghrelin

(AG) composes the remaining of total ghrelin (Delhanty et al., 2011). Originally, ghrelin was

discovered as the natural ligand for the growth hormone secretagogue receptor (GHSR), an

orphan receptor known to bind drugs that increased the release o f growth hormone (GH) from

the pituitary gland (Howard et al., 1996; Kojima et al., 2000). As such, ghrelin was found to

6

increase GH, although soon it was reported that exogenous administration of ghrelin increased

food intake and adiposity in animals and humans (Tschop et al., 2000). However, in order for

ghrelin to produce any o f its characterized effects, a modification in the 3 rd serine residue needs

to occur. This modification requires that the n-Octanoic acid fatty acid be ligated to the third

aminoacid of its molecule (serine) via acylation. This process is mediated by the Ghrelin O-

acyltransferase (GOAT) enzyme (Yang, Brown, Liang, Grishin and Goldstein, 2008). Once this

modification occurs, ghrelin is able to bind to its receptor. The non-acylated ghrelin also has

biological activity although it does not bind to GHSR (Yang et al., 2008).From an evolutionary

standpoint, ghrelin’s actions are said to help efficiently secure caloric-dense tasty foods to

protect during periods of food scarcity (Chuang, Perello, Osbome-Lawrence, Savitt, Lutter &

Zigman, 2011; Delhanty et al., 2011). Interestingly, this may also shed light on why levels o f

AG and GH are elevated within anorexic patients, while basal plasma ghrelin levels are usually

lower in obesity; possibly a counter-regulatory response to reestablish ideal body weight

(Tschop, Devanarayan, Weywer, Tataranni, Rayussin & Heiman, 2001). Ghrelin also increases

the circulating levels of prolactin, ACTH and cortisol (Nakazato, et al., 2001).

The effect o f ghrelin on different organs has been reviewed in a number o f excellent

papers, but for the present paper we will discuss the effects o f ghrelin of GHSR located in a

number of brain regions, although primarily in the control center o f the ARC.

Ghrelin receptor mediates ghrelin’s multiple functions.

Ghrelin produces its effects via its actions on the GHSR. This receptor is found in

numerous tissues including adipose tissues, liver, pancreas, pituitary gland and brain. GHS-Rla

is among the G-protein couple receptor family with the classification of transmembrane

receptors. It is composed of 366 amino acids with seven transmembrane alpha helices. This

7

subset is the functional form of the receptor. The effects o f ghrelin on growth hormone release

are mediated by ghrelin binding to the third transmembrane domain of the receptor in pituitary

gland cells (Congreve M. et al., 2010) although feeding responses may no be mediated by ghrelin

binding at this region. Based on affinity and specificity, ghrelin binds to GHSR-la and this

extracellular binding activity coordinates the specific activation of an associated G-protein that in

turn, signals downstream cascades. In short, According to Albert et al., (2009), acylated ghrelin

signals through G alpha q, i.e. diacylglycerol (DAG) and Inositol trisphosphate dP3)

intracellular messengers to create a calcium influx which is an important element in the process

of neurotransmitter activation and release.

The orexigenic and adipogenic effects o f ghrelin appear to be mediated by ghrelin

receptors in the brain. Evidence for this comes from experiments where ghrelin was delivered

chronically into the cerebral ventricles o f rats and mice using osmotic minipumps at doses that

were too low to produce peripheral effects (Tschop et al., 2000). These animals showed a

transient increase in food intake, but showed a sustained increase in weight gain and this weight

gain was attributed to an increase of adipose tissue and not in muscle or bone, and independent

of the effects o f ghrelin on growth hormone (Tschop, 2000). In fact their studies showed that the

effects of ghrelin on food intake were mediated in part through the stimulation of both NPY and

AgRP cells, because mice with mutations to these genes did not show feeding or adipogenic

responses to exogenous ghrelin infusions (Tschop et al 2000; Nakazato et al., 2001). More

evidence of ghrelin’s role in regulating metabolism via actions on the brain was demonstrated

when Theander-Carrillo et al., (2006) reported that chronic infusion of ghrelin into central

nervous system increased lipogenesis and inhibited lipid oxidation in white adipose tissue. As a

whole, these findings suggest central ghrelin infusion favors partitioning nutrients towards fat

8

storage instead of an increase in glucose and triglyceride uptake, and as a result, adiposity and

weight gain occur. Briefly said, ghrelin’s modulation within the hypothalamus is exerting

orexigenic effects on neuropeptide Y and melanocortin neurons via binding to GHSR (Cowley et

al., 2003).

Theoretical explanations on ghrelin’s pathway to communicate with Central Nervous System.

The mechanisms by which ghrelin is transported to the Central Nervous System (CNS)

remain largely unknown There are three potential theories that exist to explain the process

through which systemic ghrelin is transported across the blood brain barrier. One possibility

involves ghrelin molecules infiltrating the blood brain barrier via passive transport given the free

fatty acid that is acylated to the serine, and which could facilitate entrance to the brain because it

is lipophilic. There are, however, no data as o f yet to fully support this hypothesis. In once

particular study, it was observed in a mouse model that acylated ghrelin is readily transported

across the blood-brain barrier in the brain-to-blood direction, but the quantity o f its transport in

the blood-to brain direction appears to be negligible (Banks et al., 2002). Nevertheless in 2006,

Diano et al., (2006) observed within 10-15 minutes o f peripheral infusion, ghrelin crossed the

blood brain barrier and reach regions like the hippocampus.

The second possibility of ghrelin’s path to the brain is via its direct actions on the vagus

nerve and its brain stem afferents, which project to other brain regions like the hypothalamus.

One study revealed that vagotomy prevents peripheral ghrelin’s effect on the hypothalamus

(Date et al., 2002). However, other findings have suggested no association between ghrelin’s

effects on the brain via the vagus nerve (Tschop et al., 2000; Nakazato et al., 2001). Evidently,

the pathway in which ghrelin is transported to and cross the blood-brain barrier into the CNS

remains to be clarified.

9

Finally the third theory posts ghrelin being produced within the hypothalamus. Indeed the

ghrelin peptide has been detected in the hypothalamus of mice, rats and humans, although in

much smaller concentrations than in blood (Cowley MA et al. (2003). Ghrelin producing cells

have been found in the hypothalamus as well as in the stomach (Kojima et al., 1999). Moreover,

intracerebroventricular (ICV) and intraperitoneal (ip) injections of ghrelin produced positive

effects for GH secretion and food intake in rats fed standard chow. However, once the diet was

replaced with high-fat diet (HFD), ICV or IP injections o f ghrelin failed to induce food intake.

This behavioral data was supported with a decrease in NPY AgRP mRNA expression and failure

to stimulate growth hormone secretion following ghrelin ICV injections. These results suggest

that in the context of HFD, this particular diet influences ghrelin’s neural targets, by making

them less responsive to ghrelin, even if they are possibly produced within the hypothalamus.

More research is necessary to fully understand whether or not this theory of hypothalamic -

ghrelin-producing cells lies true. Since, the physiological role of endogenous ghrelin produced in

the hypothalamus remains undetermined, this topic remains hotly debated. Although if true,

brain derived ghrelin would pose for a novel hypothalamic circuit regulating energy homeostasis.

In any case, once ghrelin reaches the hypothalamus, DIO has displayed an impact on the

brain’s ability to respond to ghrelin, therefore creating a ghrelin resistance, which may possibly

act as a mechanism to adapt in positive energy balance.

GHSR central expression and ghrelin’s actions within these regions.

Not surprisingly, the hypothalamus and pituitary gland are the main target for circulating

ghrelin, given the ability o f ghrelin in regulating GH, prolactin and adrenocorticotropin hormone

ACTH secretion (Broglio et al., 2002). The ARC arguably contains the highest concentration and

density expression of GHSR, coinciding with ghrelin’s effects on regulating energy homeostasis,

10

although other nuclei known for their secondary role in the regulation of energy balance and

food intake also express the GHSR (Zigman, Jones, Lee, Saper & Elmquist, 2006). The pituitary

gland, second to the ARC, comprises the second highest concentration of GHSR expression. This

dense GHSR expression location makes logical sense, since it plays an integral role with GH

secretion. Within the ARC, the GHSR is selectively expressed in neurons co-expressing

neuropeptide Y (NPY) and agouti-related protein (AgRP). Activation of neurons that co-express

NPY and AgRP by centrally administered ghrelin reduces hypothalamic melanocortin tone by

increasing the inverse agonist effect o f AgRP on hypothalamic melanocortin receptors (Tschop

et al., 2000).

As described above, ghrelin increases the activity o f orexigenic peptides such as NPY,

AGRP and other orexigenic second order neurons such as orexin/hypocretin from the lateral

hypothalamus promoting anabolic processes. Ghrelin may also modulate POMC secretion

indirectly to achieve the same functions. For example, the inhibitory neurotransmitter GABA is

released locally by NPY/AGRP neurons to inhibit POMC secreting cells. Ghrelin binds to

GHSR’s to increase the activity of local NPY/AGRP axons to increase the rate of secretion of

GABA and ultimately reducing the activity o f postsynaptic POMC cells (Schwarts et al., 2000;

Horvath et al., 2001). Beyond its effects on neuropeptide secretion, ghrelin was also reported to

change synaptic plasticity within the ARC. In fact, ghrelin increases the number o f stimulatory

synapses on neurons that co-express NPY and AgRP and, concurrently increases the number of

inhibitory synapses on neurons that secrete pro-opiomelanocortin hormone from neurons that

express POMC (Abiziad, 2008; Morton, Ladenheim & Schwartz, 2001; Horvath et al., 2001).

The overall result of these changes is that ARC NPY/ AGRP neurons are more likely to be

stimulated (i.e. lower threshold) by any given stimulus, whereas POMC neurons are less likely to

11

be stimulated by any given stimulus when ghrelin levels are elevated (Morton et al., 2001; Pinto

et al, 2003).

In addition to the hypothalamus, substantial levels o f GHSR mRNA expression have

been detected in other regions that are important and work in parallel with the energy

homeostasis center. The hippocampus, in particularly the CA2 and CA3 regions o f Ammon’s

horn and the dentate gyrus express high density o f GHSR while moderate expressions were

found in the CA1 region (Diano, et al., 2006; Guan et al., 1997). The hippocampus has been

linked to cognitive processes including regulation o f the stress axis, memory formation and

consolidation, spatial memory, and navigation (Guan et al., 1997). Peripherally administered

ghrelin has been shown to reach the hippocampus within minutes and to produce changes of

synaptic plasticity correlating with enhanced spatial learning and memory (Diano et al., 2006).

Ghrelin induced plasticity was reflected in increased spine density on hippocampal dendrites as

well as in a higher number o f synapses in these spines (Diano et al., 2006). This ghrelin function

might aid as a potential mechanism to sharpen senses during food deprivation and efficiently

remember the localization of food sources and its associated positive or negative experiences.

Another brain region that contains ghrelin receptors outside the hypothalamus and may

also contribute to physiological regulating appetite actions of ghrelin is found in the caudal brain

stem. Through in situ hybridization, Zigman et al., (2006) validated the presence o f GHSR

mRNA within all three components of the dorsal vagal complex, including the nucleus o f the

solitary tract, the area postrema, and the dorsal motor nucleus of the vagus. The presence of

GHSR in these structures suggests ghrelin may manifest its actions on energy control through

this ascending pathway in parallel or complementary to the hypothalamus. Fittingly, the DVC

also contains receptors responsive to other blood-borne signals correlated with the animal’ s

12

metabolic status such as leptin receptors initiating satiety and detection of changes in circulating

glucose level (Grill, Schwartz, Kaplan, Foxhall, Breininger & Baskin, 2002).

Two of DVC’s component structures, the nucleus tractus solitarius (NTS) and area

postrema (AP), are the first to receive visceral afferent (ghrelin, leptin, insulin, cholecystokinin)

since they lie outside of the blood brain barrier. These messages subsequently drive a number of

autonomic reflexes through communications via efferent projections to hypothalamic structures

that are able to orchestrate appropriate responses (Hou et al., 2006; Hintmarch et al., 2011).

The nucleus of the solitary tract is the primary site through which gastrointestinal (GI)

afferent information enters the brain (Moran et al., 2001). It is a region that controls sympathetic

outputs and that receives information from the vagus nerve, and is also sensitive to ghrelin and

satiety signals (Date Y et al. 2002). Intra-cerebral injections o f des-acyl ghrelin performed by

Tsubota et al., (2005) within the NTS significantly reduced mean arterial pressure and heart rate.

The hypotensive activity suggests that des-acyl ghrelin contributes to the regulation of

cardiovascular control.

Ghrelin has been shown to also act upon GHSR within the NTS in the brain stem and

influence noradrenaline (NA) cell groups and project to alphal and beta2 noradrenergic receptors

in the ARC to stimulate food intake (Date et al., 2006). Cui, Li and Appleyard, (2011) also

recognized the importance of other A2/C2 catecholamine (CA) neurons in the NTS with regards

to food intake and ghrelin’s modulating effects on this neurons within energy homeostatic

function.

The area postrema (AP) also displays moderate expressions o f GHSR cells (Zigman et

al., 2006) and responds very quickly to changes in glucose, fatty acid concentrations and gut-

peptide signals in order to maintain homeostasis. Fry et al., (2008) have examined the ability of

13

ghrelin to regulate and modulate the electrical activity of area postrema neurons using voltage-

clamp electrophysiology recordings and found activation within GHSR cells of the AP,

stimulating appetite and regulating pancreatic protein secretion.

The dorsal motor nucleus of the vagus (DMNV) is located adjacent to the fourth ventricle

and serves parasympathetic vagal function including the gastrointestinal tract, lungs, smooth

muscle and heart (Niedringhaus, Jackson, Evans, Verbalis, Gillis & Sahibzada, 2008).

Sometimes called “cardioinhibitory center”, one of its roles involves slowing the heart rate.

Mediated by efferent vagus nerves, stimulation of the intermediate and rostral DMV increase

fundus synthesis o f ghrelin while stimulation of the caudal DMV caused a decrease in fundus

tone (Niedringhaus et al., 2008). This relationship may shed more light on the fundus’s role in

secreting ghrelin and its communication with the brain stem.

Faulconbridge et al., (2003) administered ghrelin to the fourth ventricle near the

brainstem to explore the functional importance of the brainstem GHSRs and compare feeding

responses with those obtained when ghrelin was delivered near hypothalamic sites. Previously

Wren et al. discovered the lowest effective ghrelin intracerbroventricular dose to induce

hyperphagic behaviors within the third ventricle near the ARC was 30 pmol. Surprisingly,

Faulconbridge et al., (2003) obtained a similar response within the fourth ventricle, near the

DVC with only 10 pmol. This suggests a possibly more sensitive area to ghrelin administration

acting on GHSRs in the DVC. Behavioral hyperphagic measures to support these orexigenic

effects included reduction in the latency to feed and an increase in the number o f meals taken

during the first few hours after treatment (Falconbridge et al., 2003). These results are consistent

with a role for central ghrelin signaling in meal initiation. In addition, these findings also draw

attention to the importance of DVC as an initial site for ghrelin within the brain. It equally

14

displays a functional parallel between the DVC and the hypothalamic arcuate nucleus and

highlights the importance of the caudal brainstem in regulating food intake by integrating

information from visceral organs and in turn conveying them to appropriate higher-order regions.

Moreover, these studies also reiterate ghrelin’s key role in modulating feeding responses within

these regions.

Finally, the mid brain ventral tegmental area (VTA), a brain region well known for its

role in the regulation of reward seeking behaviors and motivation for food also contains a

relatively high concentration o f GHSR. This GHSR expression mediates the direct interaction of

ghrelin’s effects within a subset of VTA neurons that produce dopamine (DA) and that are

associated with reward seeking mechanisms (Guan et al., 1997). Therefore, in addition to

moderating normal eating habits, ghrelin acts on the brain’s reward centers. This mesolimbic

reward circuit is important to consider when discussing energy homeostasis because it targets a

motivational reward aspect of feeding that is considered by some; the root o f our overeating

epidemic.

The VTA reward pathway is separate from the homeostatic circuit found within the

hypothalamus. These two regions often communicate whereby the VTA receives information

from the primary hypothalamus region. Depending on the energy balance status of the organism,

the VTA receives input to modulate motivational aspects o f feeding such the “liking “ and

“wanting” behaviors necessary to seek and obtain food (Abizaid et al 2009; Berridge, 1996). In

addition, being sensitive to direct ghrelin stimulation, VTA dopamine cells are innervated by

lateral hypothalamic hypocretin/orexin neurons, which are also sensitive to ghrelin (Abizaid,

2009; Toshinai, et al., 2003). Shimbara et al., (2004) reported ghrelin selectively stimulated the

intake of foods high in fat. Some research suggests these reward pathways may even override the

15

hypothalamic homeostatic centers and produce increased appetite (Grill et al., 2001). GHSR

mRNA transcript and protein are expressed in relatively high quantities within the VTA and

adjacent substantia nigra (SN) regions associated with goal directed behavior. When ghrelin is

administered centrally into the VTA and SN or into the Nucleus Accumbens (Nac), food intake

increases significantly (Naleid, 2005; Abizaid et al., 2006). The mechanisms underlying these

feeding responses involve the direct action of ghrelin on VTA DA cells as DA neurons increase

their activity in response to ghrelin as shown in experiments recording from DA cells in slice

preparations (Abizaid et al, 2006). Ghrelin does this by binding to DA cells and increasing their

firing rate or action potentials. Subsequently, ghrelin increases the release of DA into the nucleus

accumbens in association with increased feeding responses and increased motivation (Abizaid et

al., 2006; Mathon, et al., 2005). Motivation was measured via goal-oriented behaviors including

operant responses or increased locomotor food anticipation (Abizaid et al., 2006; Blum et al.,

2009; King et al, 2011). Collectively, these findings suggest ghrelin may act on the VTA to

increase food intake by enhancing the incentive value of foods (Abizaid, 2009).

1.4 Previous ghrelin ghrl/GHSR KO mice findings

Understanding the biological effects o f ghrelin using animals with single gene mutations to

ghrelin related genes.

Before discussing data associated with the use of animals with targeted deletions o f the

ghrelin gene, one needs to consider the usefulness o f genetic approaches to studying obesity. It is

clear that there are genes that are directly implicated in the development o f obesity. For example,

single mutations to the gene encoding the leptin, POMC or the melanocortin 4 receptor (MC4R)

proteins lead to morbid obesity and metabolic disorders in rodents and humans (O’Rahilly,

Farooqi, Yeo & Challis, 2003). This obese phenotype can be corrected with treatment that

16

restores the levels of these proteins. Moreover, climbing rates of obesity correlate well with the

sedentary lifestyle of human populations around the world and the increase in the availability,

amount and affordability o f high calorie meals and snacks. Thus, an interaction of genetic make­

up, a decrease in physical activity, and an increase in the intake of calories are at the root o f the

obesity pandemic.

A number o f genetic alterations have been shown to predispose some individuals to gain

more weight than others given similar lifestyles. Thus, excessive high-caloric food consumption

coupled with insufficient physical compensatory activity may impact metabolism in those who

are genetically predisposed to maximize metabolic efficiency and fat storage (Delhanty et al.,

2011). An example of this are individuals with haplo-insufficiency for Sim 1 (a transcription

factor required for normal hypothalamic development) (Holder, Brutte & Zinn, 2000), or for

brain-derived neurotrophic factor (BDNF) (Gray et al., 2006) have been shown consistently to

become more susceptible to obesity. Similarly, heterozygous loss-of-function missense mutation

to the Trk B BDNF receptor are also more vulnerable to become obese compared to non-mutant

individuals (Gray et al., 2006).

Finally, a good body of evidence has shown one genetic variant termed Fat mass and

obesity-associated protein (FTO) predisposes obesity by affecting the same homeostasis pathway

whereby its expression is altered during fasting and feeding (Dina et al, 2007). Individuals who

are homozygous for the high-risk allele (AA) of FTO weigh on average 3 kg more than

individuals with two low-risk alleles, with heterozygotes having an intermediate risk (Farooqi &

O’Rahilly, 2006).

In all, while there are a number o f genes that are clearly implicated in the regulation of

energy balance, and their mutation can lead to obesity. It is likely that human obesity comes as a

17

result of the interaction between genes and environment, and that while some may be

predisposed to gain weight, they may not because their energy expenditure exceeds their caloric

intake (Kanasaki et al., 2010). In contrast, others may gain weight in spite o f increased energy

expenditure and decreased caloric intake because of their genetic make-up resists a weight-loss

phenotype.

Some have suggested that individuals such as these are the product of evolutionary

pressures where an energy "thrifty" set of genes allowed our ancestors to adapt and survive

periods where food was scarce such as winter. This changed with the revolution in our modem

society’s food industry, and those genes that helped our ancestors survive are now the ones that

are decreasing life expectancy because of health complications associated with obesity (Kanasaki

et al., 2010).

Diet-Induced Obesity Mice Models

Current animal models of obesity typically include mice that have natural or genetically

engineered mutations to single genes (Appendix A). In addition, there are models that include

selective breeding for obesity or obesity resistance, both of which are presumed to be the result

of alterations in several genes. Finally there are models where animals are exposed to high

caloric diets or to varied choices o f palatable diets. Both of these experimental manipulations are

referred to as diet induced models of obesity.

Animal models representative o f Diet-Induced Obesity often include a High-Fat Diet

whereby different strains acquire different responses to the HFD. Some inbred strains o f mice

exhibit significant obesity when fed HFD while other remain lean (West et al., 1992). Among the

mice list prone to obesity are the C57BL/6J mice, sand mice and spiny mice because they display

similar abnormalities that humans experience during high-fat diets (Collins et al., 2004).

18

There are many examples of natural monogenetic (single gene) mutations that lead to

obesity and metabolic disorders like Type II diabetes. Examples o f these are rodents with

mutations to the leptin or leptin receptor gene. The first ob/ob mouse arose by chance in a colony

at the Jackson Laboratory in 1949 (Ingalls, Dickie and Snell, 1950). These mutants were first

described in the 1950's in mice colonies breeding the C57BL/J6 mice strain. These mice were

originally termed as ob/ob, and their discovery was followed by the discovery of another strain

of obese mice that arose spontaneously in the same colony and whose metabolic phenotype was

similar to the ob/ob mice strain with an exaggerated diabetic phenotype. These mice were termed

as db/db mice (Coleman, 1978; Friedman & Halaas, 1998). The genes that were mutated in these

animals were the leptin and the leptin receptor genes respectively, and the identification of these

mutated genes and the proteins encoded by these genes are considered one o f the most

significant findings with regards to energy balance regulation (Friedman et al., 1998).

Similar mutations have been identified in rats where the most common models of obesity

is the Zucker Fatty Rat (ZFR), a strain of rats that emerged from the Long Evans rat strain and

one where the protein encoded by the leptin receptor gene is altered, and therefore has decreased

leptin signaling properties (Kurts, Morris and Pershadsinghl989). These rats display an early

onset to obesity at 5 weeks of age, although they do not go on to develop diabetes (Bray et al.,

1977). Only a sub-strain of ZFR termed Zucker diabetic fatty (ZDF) has been found to develop

diabetes (Friedman et al., 1991) and used in diabetes animal models. Another monogenic mouse

obesity model is the lethal yellow Agouti mouse, which exhibits a mutation on the gene that

encodes the agouti related peptide (AgRP) (Bultman et al., 1992). This was the first obesity gene

characterized as the molecular level and is now known to be critical for the regulation of energy

balance (Abizaid et al., 2008, Bultman et al., 1992). This agouti gene expression is also found in

19

human adipose tissue; hence it’s relevant nature to human obesity (Kwon, Bultman & Loffler;

1994). In terms of diet-induced obesity rat models, the Sprague Dawley and long Evans rats are

commonly used as models for HFD-induced obesity (Srinivasan & Ramarao; 2007) mostly

because these strains easily gain weight compared to other rat strains.

Monogenic models provide important biology findings of obesity while polygenic models

illustrate the mediation of obesity through the interaction between genes and environment.

Indeed, while there are many rodent strains that become obese when exposed to a high-fat diet,

not all inbred strains will develop obesity. These findings suggest potential genotypes that

predispose animals to develop obesity while others might resist obesity (West et al., 1992).

Indeed there are strains of rats originating from the common Long Evans strain that are either

prone or resistant to developing obesity when fed a high fat diet. Levin et al., (2003) compared

leptin, insulin and ghrelin levels, using radioimmunoassay kits, between rats that were bred to be

prone to diet-induced obesity (DIO) and to rats bred to become diet resistant (DR). Levin et al.,

(2003) discovered a significant increase in body weight between 5 and 7 weeks of age relative to

their WT littermates, when fed ad libitum standard chow. During the 5th week period, DIO

prone rats consumed 9% more of chow than DR and as a result, gained 19% more body weight.

Ghrelin levels also seem to differ during the onset o f the dark period between the two groups

whereby DIO displayed 29% lower plasma ghrelin levels. Despite these significant changes in

behavioral measures and ghrelin, no differences in leptin and insulin were reported. When

animals were acutely exposed to a HFD (HE; 31% fat) at 6 weeks, previous behavioral measures

on the standard chow were amplified. DIO rats were reported to consume 70% more HFD and

contrary to the standard chow diet, exhibited significantly higher insulin and leptin levels than

DR rats. These data suggest a critical period in development were rats are vulnerable to develop

20

abnormal regulation of appetitive hormones such as ghrelin, leptin and insulin. Subsequently,

rats prone to DIO are more likely to significantly increase appetite and body weight during this

critical period. Some ghrelin deletion studies echo similar results during this critical 5-7 week

period of development (Cowley et al., 2003; Sun et al., 2004).

A recent study conducted by Chen, Wang, et al., (2012) examined the long-term effects

of different macronutrient diets including both high-fat diet and high-protein diet in Sprague-

Dawley rats. Pregnant or lactating females were fed either one of these diets or standard chow

and offspring continued these same diets post-weaning. Plasma ghrelin, insulin and glucose

measures were investigated at several time points in the off-springs development including the

first, third, seventh, and tenth postnatal days (Chen et al., 2012). Also, these same tests were

repeated on the same animals towards the end of second, third, fourth, eighth, and twelfth week

(Chen et al., 2012). In accordance with previous studies, rats in all three groups exhibited a

negative correlation between ghrelin and glucose level. However, only within the high-fat and

high-energy group, did rats display a negative correlation between plasma ghrelin and plasma

insulin relative to WT rats (Chen et al., 2012). Hence, long-term diets including high fat or high-

protein content both negatively affect the relationship between insulin and ghrelin expression

within the plasma.

Other models include; the New Zealand Obese (NZO) mouse strain which represents a

good model of hyperphagia and reduced energy expenditure, the Tsumura Suzuki Obese

Diabetes (TSOD) Mouse which become obese despite not developing diabetes because of their

increased beta cell mass and good insulin maintenance secretion (Iiuzuka, et al., 2005), the M l6

mouse which exhibit hyperphagia, hyperinsulinemia, and hyperleptinemia compared to controls

(Allan et al., 2004) and finally, the Kuo Kondo (KK) mouse which is widely used for testing

21

experimental therapies in obesity and diabetes research (Okazaki et al., 2002). Finally, Otsuka

Long Evans Tokushima Fatty (OLETF) Rats established in Japan became an obesity and

diabetes model. Males tend to display hyperplasia o f pancreatic islets at 25 weeks of ages and at

60 weeks of age, islets become atrophic (Kawano et al., 1994).

Ghrelin and Ghrelin Receptor mutant mice

A number of genetically engineered rodent models have emerged in the past 10 years to

better understand the physiological significance of the ghrl/GHSR system. These models have

been tremendously useful, although they have a number of limitations. The first transgenic

rodent used for the study of ghrelin was a genetically modified rat with an antisense sequence

aimed at reducing GHSR expression that was under the control o f the tyrosine hydroxylase

promoter (Shuto et al., 2002). Western blot and immunocytochemistry analysis were performed

to confirm lower GHSR protein levels in the ARC within transgenic (Tg) rats in comparison to

non-Tg littermates. The tyrosine hydroxylase (TH) promoter is usually active within GHRH-

containing neurons in the Arc (Niimi et al., 1992). Therefore, the aim of this study was to

disrupt the function of GHSR within the Arc and discover the function of the GHSR gene. Shuto

et al., (2002) reported that these transgenic rats had in fact, lower body weight and less adipose

tissue within relative to WT controls.

Soon after, several reports were published describing the phenotype of mice with genetic

mutations to the ghrelin and the GHSR gene (Appendice 1). There were three labs that generated

these lines o f mice independently (Sun et al 2003; Wortley et al., 2004; Zigman et al., 2005).

While these mice were generated in different ways, they all shared a common phenotype under

normal conditions (Sun et al., 2008). The phenotype of both GHSR and ghrelin KO mice was

surprisingly similar to that of their WT littermates (Sun et al., 2008). However. Ghrl and GHSR

22

KO mice did show resistance to gaining weight under a high fat diet regimen, and showed less

adiposity in body composition under normal conditions (Zigman et al., 2005). Moreover, GHSR

and Ghrl KO mice displayed differences in spontaneous locomotor activity compared to their

WT littermates (Blum et al., 2009). Both of these genotypes are enhanced in mice with mutations

to both the ghrl and the GHSR gene. When animals are exposed to negative energy balance,

GHSR KO displayed lower glucose levels suggesting better insulin efficiency and glucose

clearance (Sun et al., 2006; Cowley et al., 2005). Interestingly, the offspring of GHSR KO mice

mixed with leptin deficient Ob/Ob mice, while still overweight, showed improvements in

glucose homeostasis. The fact that the GHSR and ghrelin KO mice failed to produce a clear-cut

metabolic profile tended to discourage ghrelin research. Some neuroscientist came to the

conclusion that ghrl was a redundant mechanism within appetite regulation (Sun et al., 2003;

Wortley et al., 2004). Nevertheless, other scientists such as Zigman et al. (2005) persisted to

generate their own GHSR KO mice and discovered otherwise. Perhaps some explanations derive

from the various mice strains generated throughout those transgenic mice studies and the ways in

which the deletion was manifested. Other supplementary reasons could include the different

concentration of fats within the HFD mice were subjected to as well as the critical age period in

which they were initially exposed to HFD. Also for these inconsistent results, it is possible that

these mice, given their lack of ghrelin throughout their life span, developed compensatory

mechanisms that allow them to deal with the lack of ghrelin or its receptor without obvious

challenges. In fact this has been found to be the case with mice that mutations to the NPY or the

AgRP genes (Higushi, Niki and Shiiya; 2008). When mice were kockouts from birth, reports

suggest no particular distinguishable phenotype. However, when mice are manipulated within

adulthood, these mice without NPY/AgRP display a strong anorexic phenotype, at times fatal.

23

Despite these compensatory mechanisms, certain defects have been reported in GHSR and Ghrl

KO mice and these include deficits in motivated behaviors for food (Nogueiras, Tschop &

Zigman; 2008), manifested through reduced locomotor responses to scheduled meals (Blum et

al., 2009; Abizaid; 2006), reduced locomotor responses to psychoactive drugs like cocaine or

nicotine, and reduced intake of alcohol (Clifford, Rodriguez, Schul, Hughes, Kniffin, Hart, Etain,

Brunei, Fehrentz, Martinez and Wellman; 2011). In addition, these GHSR KO Ghrl KO mice

appear to have learning and memory deficits (where ghrelin typically neuroprotects cognitive

functions), altered responses will resume once subjected to acute stress. However, they are

reported to become prone to depressive like states following chronic stress (Patterson et al.,

2010).

15 Hypothesis: Validating the GHSR KO DIO Rat Model

GHSR KO rat model.

One of the disadvantages working with mice is that their behavior is more difficult to

examine than that o f rats. In general most behavioral tasks with mice require substantial amount

of training, handling, and in some cases tasks that work well in rats are not ethologically valid in

mice. This has posed a number of limitations to the studies o f behavior in mice with genetic

deletions to the ghrelin and ghrelin receptor gene. Most notably, the ability for rats to learn and

perform more complex behavior tasks is significantly greater than that in mice.

Recently, a rat strain with a mutation to the promoter region of the GHSR gene became

available commercially. The availability of this strain has been met with enthusiasm by several

labs and there are two early reports showing that these rats, like ghrl and GHSR KO mice, have

attenuated responses to cocaine and nicotine (Clifford et al., 2011 ;Wellman, et al., 2011). As

24

expected these rats do not show feeding responses to ghrelin treatment. Nevertheless, this GHSR

KO rat line and its background strain remain poorly characterized.

Given the phenotype of the background Fawn-Hooded Hypertensive (FHH) strain, it is

uncertain what kinds of metabolic responses exist in FHH-GhsrmlMcw‘(see methods for details).

The present study was conducted to examine the metabolic profile o f FHH-GhsrmlMcwi in

comparison with that o f FHH wild type rats under normal conditions and following exposure to

changes in food availability, and after prolonged exposure to a high fat diet. We hypothesized

that, like GHSR KO mice, rats with a mutation to the GHSR would eat less and show attenuated

feeding responses to a fast than their WT counterparts, and that they would gain less weight and

show altered glycemic responses to a glucose challenge. Finally we expected that these GHSR

KO rats would show altered hypothalamic expression of peptides o f the melanocortin system and

circulating metabolic hormones promoting a leaner phenotype than WT rats. To test this

hypothesis, we conducted 3 behavioral experiments and analyzed 2 further experiments

following sacrifice. The experimental procedure is illustrated in Figure 1.

2 Methods

2.1 Fawn Hooded Rat Background Strain

As mentioned, the background strain that was used to generate this rat model is fairly

uncommon. In this case, the strain chosen was the fawn hooded rat, a line established as an

outbred stock in Kroning, Gottingen from rats o f unknown antecedents (although most likely

Wistar and Long-Evans). Following a couple displacements, they finally were established in

Hannover, Germany in 1968 (F I6). They remained outbred until the mid-1980s when the colony

was relocated to Erasmus University in Rotterdam, Netherlands. At this point, Dr. A.P. Provoost

generated the standard brother x sister inbred generations. A minimum of 20 brother x sister

25

generations is necessary to obtain a level of residual heterozygosity that is on average less or

equal to 0.01 (Green, 1981). From this process, two strains emerged. One became known as the

fawn hooded hypertensive rat (FHH) where the other was labeled fawn hooded low blood

pressure (FHL).

It was not until the 1990s that the FHH rat made its way to the Medical College of

Wisconsin for future North American studies. Dr. Provoosfs strain was used to develop a panel

of consomic rats with FHH as the background strain and Brown Norway chromosomes

introgressed into the FHH background. From this background, mutant strains were developed

primarily using N-ethyl-N-nitrosourea (ENU) mutagenesis where male founders are injected.

This chemical is an alkylating agent and is a highly potent mutagen. It essentially manifests its

functions by targeting spermatogonial stem cells found in mature sperm. (Ethylnitrosourea-

Compound Summary; National Center for Biotechnology Information, 2012). For the GHSR

gene, ENU can induce 1 new mutation in every 700 gametes. Once bred with females, the pups

are controlled and screened to detect and confirm the proper GHSR gene was in fact targeted to

possess ENU-induced mutations. This is conducted using an enzyme-based heteroduplex

cleavage assay termed TILLING assay (Medical College of Wisconsin). Both the GHSR KO and

wild type FHH rats were developed using ENU mutagenesis. Medical College of Wisconsin

happens to be the supplier for Biotransposagen pharmaceuticals service in Kentucky, USA, and

the source for the animals in our particular study.

The full sub-strain abbreviation of fawn hooded hypertensive rats begins with the core

symbol o f the original strain followed by an abbreviation o f where the strain is allocated. These

two subsets of information are separated by a forward slash (e.g., FHH/EurMcwi; substrain

derived at Medical College of Wisconsin Institute (Mcwi) from the colony maintained in Europe.

26

According to the Rat Genome Database, the symbol representing our particular GHSR KO

mutation includes FHH-GhsrmlMcwi to represent mutation 1. This mutation generated by ENU

occurs in the codon CAG/TAG, which changes the AA q343Stop o f the GHSR gene (Rat

Genome Database, 2012).

The FHH background strain is known for developing hypertension and kidney

complications in late adulthood and generally has a shorter life span than most common

laboratory rats strains. These consequences ultimately lead to shorter lifespan and may include

focal and segmental glomerular sclerosis, pulmonary and systemic hypertension and proteinuria

(excessive serum protein levels in urine) at a young age.

FHH rats also tend to express a platelet storage pool deficiency due to the red-eyed yellow allele

r (Suckow et al., 2006). Platelets aid the formation o f blood clots and prevent excessive bleeding

to occur by coagulating. Platelets also release several growth factors, which play a role in

regenerating connective tissues. Therefore, when platelets are forcibly excreted from the

circulation quicker than the bone marrow’s production, thrombocytopenia occurs. Because FHH

rats develop dysfunction in filtering blood to form urine, spontaneous or excessive bleeding

arises. Furthermore, Margo et al., (1986) measured the differences in urine secretion between

hypertensive fawn hooded rats in comparison to Wistar rats. Margo and colleagues found a

significantly greater amount of catecholamines within the urine o f FHH rats. With the

administration of an antihypertensive (debrisoquin sulfate) compound, FHH rats blood pressure

diminished to normal levels and their catecholamine excretion significantly decreased.

Altogether, these findings support a link between higher sympathetic output and hypertension

within FHH rat strain suggesting a response that acts primarily on the cardiovascular system

27

(increased heart rate), mediated indirectly via catecholamines secreted from the adrenal medulla.

(Silverthom and Unglaub; 2009).

Furthermore, Tordoff et al., (2010) published an extensive observational set of

experiments deciphering tastes preferences among 14 rat strains. Rats were subjected to 17

different taste compounds and of those, FHH rats showed significant preference for water,

ethanol, saccharin, sucrose, and monosodium glutamate (MSG) (while food intake remained

similar to other groups). However, FHH rats failed to exhibit any significant differences with

citric acid, capsaicin, or com oil consumption in comparison to the other 13 strains. These

findings suggest FHH rats desire salty, sweet, alcoholic beverages and are less inclined to

consume high fat/high caloric, sour, and bitter compounds. The same study conducted by

Tordoff et al., monitored body weights for both male and female rats of all strains and recorded

FHH males the 6th lightest rat among 14 strains; weighing less than half the weight o f the heavier

Long Evans rats. This study occurred over a period of 48 weeks. Consistent with Tordoff s

study, low FHH rat body weights were also reported by Wang et al., (1988).

Additionally, Altemus et al., (1994) discovered higher amounts of freezing behavior in

response to stress within FHH rats compared to Wistar rats. Through in situ hybridization,

quantification of corticotropin releasing hormone (CRH), arginine vasopressin (AVP) and

noradrenergic stress response and arousal systems were examined in FFH rats. Subsequently,

FHH rats displayed elevated levels o f CRH mRNA in the central nucleus o f the amygdala and

lower levels of CRH mRNA in the paraventricular nucleus o f the hypothalamus. In addition to

this, FHH exhibited significantly lower adrenal weights compared to Wistar rats despite no

differences in corticosterone levels. These results suggest a depressed hypothalamic-pituitary -

28

adrenal axis activity, which may explain some of the systemic and behavioral traits these FHH

rats display.

2.2 Animal Care

Male rats with targeted mutations to the ghrelin receptor gene (GHSR KO) and their WT

littermate controls were bred at the Carleton University Neuroscience Institute animal facilities

Overall, the GHSR KO rats were seemingly of normal size and their behavior under normal

laboratory housing conditions were indistinguishable. All methods followed the guidelines o f the

Canadian Council on Animal Care and approved by Carleton University’s Animal Care

Committee. Rats were from breeding pairs originally obtained from Transposagen Bio

(Lexington, KY, USA). Animals were kept under a 12-h light/dark cycle with the onset o f light

set at 08:00 hr. In all experiment conditions, both groups were provided full access to food

unless otherwise stated. To verify genotypes and that the GHSR KO group were in fact missing

the GHS receptor function, the DNAeasy kit was conducted on adipose tissue samples from both

groups following the procedural guidelines provided by the kit and using primers detecting the

presence of the GHSR.

2.3 Generation of GHSR KO rats.

Fawn-hooded hyptemsive (FHH)-Ghsrml/Mcwi {[[GHR-R (-/-)] strain were created by the

PhysGen Program in Genomic Applications through ENU mutagenesis as described above. This

rat line was obtain from Transposagen Biopharmaceuticals, Kentucky, USA -and Medical

College of Wisconsin, USA.

2 9

2.4 Experimental Design

S t C H t

8 WT 8 KO

D evelop m en t Period

4 wks

A dulthood

15 wks

8 WT 8 KO1

Fast GTT Sac

15 wks 17 wks 18 wks 31 wks 32 wks

lI

A d Lib H ig h -F a t Diet

Figure 1. This experimental design encompasses the first three experiments. The last two

experiments involve brain and hormonal profile analyses.

Experiment 1

Differences in food intake and body weight between GHSR KO and WT rats in developmental

conditions.

In this study, a cohort of age-matched rats including GHSR KO and WT (N=16; WT

n=8, KO n=8) were placed on standard chow at 3 weeks post-weaning and remained on this diet

for 3 months. We examined food intake and body weight in GHSR KO and WT rats over this

long-term period. Both parameters were assessed 5 days/week throughout this time course. Rats

were single-housed and monitored daily for standard chow intake (Teklad Global 14% Protein

Rodent Maintenance Diet 2014; Harlan Laboratories) and body weight gain. The total caloric

content of this diet was 3.4 kc.al/g (Appendix B). These food intake and body weight

measurements were recorded daily using an electronic scale capable of measuring weights

reliably and accurately to the .lgm level (Fisher Scientific) with an error o f ± .05 gm.

30

Experiment 2

Differences in standard chow intake and body weight in adulthood

Before the diet transition to HFD, rodents were given an extended baseline o f 3 weeks on

continued standard chow where food intake and body weight was continuously measured daily.

Experiment 2.1

Overnight fasting challenge.

On the evening of day 13 of this baseline, an overnight 16 hr fast was conducted. All

animals had their food removed overnight until 8:30am on Day 14. At this time, food was

returned to their cage and the rebound feeding responses o f all rats were measured for the next

six hours (30 min, lhr, 2hr, 4hr and 6hr) to determine differences in rebound feeding between

WT and GHSR KO rats.

Experiment 3

Differences in food intake and body weight between GHSR KO and WT rats under chronic high-

fa t diet exposure in adulthood.

Following experiment 1, GHSR KO and WT FHH rats had their normal chow removed after 3

weeks and were now exposed to a high fat diet (60%cals from fat; Open Source Diets). In order

to examine the role o f GHSR on long-term body weight homeostasis, we challenged GHSR KO

rats to a western HFD for 3 months. This diet contained a total of 5.24 kcal/g o f energy density

(Appendix C). As in experiment 1, daily body weight and food intake measurements were

recorded. In addition to these parameters, on one week before sacrifice, both GHSR KO and WT

rats were subjected to a Glucose Tolerance Test.

31

Following the study, body weight composition was assessed through carcass analysis.

This enabled to determine if body weight gain was attributed to excess body fat within their body

composition.

Experiment 3.1

Glucose Tolerance Test (GTT).

As previously mentioned, a week before sacrifice, GHSR KO and WT were fasted

overnight and administered an intraperitoneal (ip) bolus injection of glucose (2g/kg) mixed in

saline to test for glucose clearance. Blood glucose was measured at intervals o f 15 min, 30 min,

60 min, and 120 min following the injection, using a Bayer glucose meter on samples obtained

from the tails of the animals using conventional needles included in the glucose testing

apparatus. Glucose tolerance was assessed by calculating the area under the curve of the glucose

excursion curves. The body weights were also measured at 24 and 48 hr after refeeding.

A week later, animals were again fasted overnight and sacrificed by rapid decapitation. At this

time, glucose was also measured to determine fasting glucose levels at time o f decapitation.

Sacrifice.

At the time of sacrifice, various tissues were obtained including brain (fast ffeezed), liver,

white adipose tissue, and muscle. Additional blood samples were also collected to measure blood

concentrations of free fatty acids and metabolic hormones like leptin, insulin, growth hormone,

PYY, and active ghrelin. For the purpose of this thesis we will only discuss the processing of

brain tissues and blood samples.

Experiment 4

Differences between GHSR KO rats and WT rats in the expression o f genes encoding fo r

hypothalamic metabolic peptides.

32

Immediately after sacrifice, the brains from all rats were removed from the skull and the

hypothalamic area were rapidly dissected and processed for RT-qPCR to observe any differences

between GHSR KO rats and WT rats within appetite regulating mRNA expression in the ARC.

Transcripts for the GAPDH and beta actin protein were used as controls. Total RNA of cells was

isolated using TRIzol Reagent (Invitrogen, Carlsbad, ca, USA), following the manufacturer’s

instructions. The cDNA was synthesized from lug RNA using the Superscript II First-Strand

Synthesis System for RT-PCR (Invitrogen). Real-time RT-PCR was performed on an ABI 7900

using the SYBR Green PCR Master Mix (Applied Biosystems, Carlsbad, CA, USA). After

amplification, the PCR product was subjected to 2% agarose gel electrophoresis. GAPDH and

beta-actin were used as internal controls. All primer and probe information are depicted in

(Appendix D). RNA was treated with DNAse and ran on the Nano drop to quantify the

concentration o f RNA in each solution sample. The ratio o f sample purity (260/280) and

spectrophotometer absorbances between 1.8 and 2.0 nm wavelengths are the accepted standards

we used for measuring purity of RNA extractions in these samples.

Experiment 5

Differences between GHSR KO rats and WT rats in and in circulating metabolic hormonal

profile.

Blood was collected after an overnight fast using EDTA-coated Microvette tubes

(Becton, Dickinson and Company (BD) and immediately chilled on ice. After 15 minutes of

centrifugation at 3000g and 4°C, plasma pipetted and stored at -80°C.

Rat Gut Hormones.

To determine the levels of metabolic hormones at the time o f death, we used Millipore’s

Milliplex Rat Gut hormone MAP Panel (Billerica, MA), a kit that allowed us to quantify many

33

gut hormones simultaneously. Our hormones of interest were insulin, leptin, pancreatic

polypeptide and total PYY found within rat plasma. Milliplex MAP is based on the Luminex

Map technology where bioassays occur on the surface of the fluorescent-coded beads termed

microspheres. After being processed by the method provided by the kit and based on the

intensity of the fluorescent dye on the reporter molecule, the program is able to identify and

quantify the amount of insulin, leptin and PYY found within the rat plasma. All samples were

measured in duplicate.

Active Ghrelin.

To selectively quantify active ghrelin in our (WT n=5, KO n=5) plasma samples, the

Millipore Rat/Mouse Ghrelin (active) ELISA kit (Billerica, MA) was conducted by the method

provided with the kit. This assay is characterized by a Sandwich ELISA type, which initially

catches the active form of ghrelin within the plasma sample with anti-human ghrelin IgG. From

the standard curve measures, we were able to calculate and identify the concentration of active

ghrelin (pg/mL) in our unknown samples. Within this particular assay, the typical amount o f

active ghrelin should range between 25pg/mL to 2,000 pg/mL. All samples used in this assay

were tested in duplicate.

Plasma Triglycerides.

In order to examine differences in plasma levels of triglycerides we used the Millipore

Adipolysis Assay kit (Billerica, MA). This is a standard method to determine triglyceride

concentrations, and it involves enzymatic hydrolysis by lipase o f the triglycerides to glycerol and

free fatty acids. The glycerol produced is then measured by coupled enzyme reactions. To

quantify the amount of triglycerides in the sample, a colorimetric or fluorometric measurement

of the glycerol released is used. A higher absorbance at 540 nm is directly proportional to

34

triglyceride concentration of the sample. Samples were assayed in duplicates.

Growth Hormone.

To detect and quantify Growth Hormone in our rat plasma samples (N=10), we used the

Rat/Mouse Growth Hormone ELISA kit from Millipore (Billerica, MA) by the method provided

with the kit. We expect to see GHSR KO with less Growth Hormone compared to WT

littermates. Growth Hormone concentrations in our plasma samples can be interpolated onto the

standard curve and analyzed.

3 Results

Experiment 1 Behavioral characteristic of the FHH rats during Developmental Period.

Food intake.

A 2X11 one-between-one-within ANOVA on rats cumulative food intake was conducted

with Genotype (GHSR KO, WT rats) as the between subjects factor and Weeks (Week 5 to

Week 15) as the within subjects factor. The results showed no main effect for genotype F (1, 14)

= .168, p > .05. There was a main effect for weeks, F (2.210,30.936) =38.989 p < .0001. There

was no genotype X week interaction, F (2.210,30.936) = 1.295, p > .05 (figure 2a).

Weeks

35

Figure 2a. Food intake measure from 4 weeks to 15 weeks of age with ad libitum standard chow

conditions.

Body Weight.

A 2X12 one-between-one-within ANOVA on rats cumulative body weight was

conducted with Genotype (GHSR KO, WT rats) as the between subjects and Weeks (Weeks 4-

15) as the within subjects factor. The results showed a significant main effect for genotype, F

(1,14) = 9.465, p = .008, and a significant main effect for weeks, F (2.412, 33.769) =2727.389, p

< .05. GHSR KO rats accumulated significantly less weight over the course o f the 11 weeks.

There was no significant genotype X week interaction, F (2.412, 33.769) =1.416, p > .05 (Figure

2b)

3&b'33^ S•5*8o + ca •Vw>rawV ><

350

300

250

200

150

100

50

0

WT

-KO

8 9 10 11 12 13 14 15W e ek s

Figure 2b. Average body weight of GHSR KO and WT rats between weeks 4 and weeks

15 old with ad libitum standard chow diet.

36

Body Weight.

Following the first 4 months, WT control FHH rats (M=204.20 SD=7.80) weighed

significantly more diet than the KO (M=189.51 SD=10.80), t (14) = 3.12 p > .05 (Figure 3) with

ad libitum standard chow (Figure 2b).

M

-eM•mm

rsoAuM«t*V><

210

205

200

S 195uVi

190

185

180

175

□ WT

■KO

Figure 2c. Body weight following 4 months post-natal with ad libitum standard chow conditions.

Experiment 2 Behavioral characteristics of the FHH rats after 4 months of age.

Reduced chow intake in GHSR KO rats.

Food intake was analyzed after once it was normalized to account for body weight

differences between the groups at the onset of 4 months of age. When normalized to body

weight, an independent groups t-test revealed that GHSR KO rats (M = 0.082, SD = 0.005) did

37

not significantly consume less chow than WT rats (M = 0.087 SD = 0.005), (Figure 3) t (14) =

1.5663, p = .11.

>.*©1 ©

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0,088

0,086

0,084

0,082

0,08

0,078

0,076

0,074

p = . l l

□ WT

■ KO

Figure 3. GHSR KO consumed the same amount o f chow as WT littermates in adulthood.

Reduced body weight o f GHSR KO rats when subjected to chow in adulthood.

In adulthood, GHSR KO demonstrated significant changes in terms of body weight.

GHSR KO (M=289 SD=16.97) weight significantly less than WT (M=307.63 SD=12.32);

(Figure 4a-4b) t (14) = 2.51 p < .05.

38

uDC ™C -Hi 4 5 IS u(fl T3

J 3 flj DC'3 s * S >, « to <2 o «*ca g

*

315310

305300

295290

285

280275

270265

□ WT

■KO

Figure 4a. Average body weight following 3 weeks of standard chow.

*o

c« as «*S— io «C 4.06 Toc

•a .sO A A c>» —13•a06reiatV><

2,5

1,5

0,5

□WT

■ KO

Figure 4b. Daily body weight gain following 3 -week standard chow diet in adulthood.

39

While on standard chow, the average daily body weight gain of these 16 rats (n=8/group)

did not differ significantly between groups although a lower body weight seems to be displayed

in the GHSR KO compared to the WT.

Experiment 2.1.GHSR KO rats eat less following an overnight fa s t

A repeated measures ANOVA was conducted to examine differences in the intake of

food shown by GHSR KO and WT controls after the animals were fasted overnight and

subsequently given free access to standard chow diet the following morning. As can be seen in

Figure 5, the periodic measurements o f food intake following the morning representation

revealed a constant lower rebound feeding within GHSR KO rats. Cumulative food intake also

suggests GHSR KO display a rebound feeding to a lesser of extent.

As shown in figure 5, the repeated measures was conducted with genotype (WT or GHSR

KO) as the between subjects factor and time (30 min, 60 min, 120 min, 4 hrs, 6 hrs) as the within

subjects factor. The results showed a significant main effect for genotype, F (1, 14) = 4.92, p <

.05, r)2 = .26 and a significant main effect for time, F (2.81, 39.36) = 45.02, p < .05, r|2 = .76. The

results of the unpaired t-tests indicated that GHSR KO (M = .02, SD =.004) consumed

significantly less g/per body weight than WT littermates (M=.03 SD =.009) at 4 hrs, t (14) =

1.93,p < .05. In terms of the genotype, GHSR KO rats ate significantly less during rebound

feeding than WT littermates. In both main effects, the null hypothesis is rejected. However, there

was no significant genotype X time interaction, F (2.81, 39.36) = .26, p > .05 ,n2 = .019. Thus,

the impact of the genotype did not depend on time.

4 0

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0,06

0,04

0,02

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□ WT

■ K O

Figure 5. Rebound feeding measures between GHSR KO and WT.

Experiment 3. Behvioral characteristics of adult WT and GHSR KO rats when chronically

subjected to HFD.

Reduced HFD intake in GHSR KO rats.

In order to examine the physiological significance of GHSR on long-term body weight

homeostasis, we challenged GHSR KO rats and wild-type littermates to a Westem-type HFD for

3 months. As done when the animals were fed regular chow, caloric intake was calculated in

proportion to body weight. When accounting for body weight, GHSR KO rats (M=0.043

SD=0.006) exposed to the HFD exhibited consumed significantly less calories than their WT

controls (M=0.039 SD=0,0008), t (14) =3,024; p < .05; (Figure 6-7).

41

>25

«

0 ,045

0,044

0,043

□ WT

■KO

- g 0,0420 faj<2 «5 0,041 ■o +.a 0,04

1 0,039h.Z 0,038

0,037

0,036

Figure 6. Accounting for body weight, GHSR KO consumed significantly less HFD.

0,21

0,205

0,2

0,195

0,19

0,185

0,18

0,175

0,17

Figure 7. Total Daily average HFD intake.

Differences between groups in body weight gain when subjected to HFD.

GHSR KO (M=64.94 SD= 19.84) displayed a slight decrease in resisting body weight

gain within the chow context compared to WT (M= 79.63 SD=19.91); (Figure 8) t (14)= 0.9901,

p = .1440.

“ «-2 m «si I£ 2 £ * s “s ' s -H

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42

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Average daily Body Weight gain during 3 months HFD (g) 1 SEM

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v:*2 .eraooOS

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Figure 8. Total average

body w

eight gain follow

ing 3

months

exposure to

HFD

.

Average total body weight gain after 3 months HFD (g) ± SEM

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Differences in body composition.

No significant differences were observed between groups in body composition comparing

fat mass between groups. As shown in figure 10, Visceral fat: WT (M= 18.50 SD= 1.67) KO

(M= 19.52 SD= 2.20) t (13) = 1.02 p > .05; Retroperitoneal fat: WT (M= 12.82 SD= 2.34) KO

(M= 12.71 SD= 1.28) t( 13) = 0.11 p > .05; Subcutaneous fat: WT (M= 23.18 SD= 3.13) KO

(M= 26.30 SD= 3.16) t (13) = 1.92 p > .05; Brown fat: WT (M=0.54 SD= 0.13) KO (M= 0.57

SD= 0.15) t (14) = 0.47 p >. 05; Total fat: WT (M=55.04 SD= 6.34) KO (M= 59.09 SD= 6.30) t

(13)= 1.24p > .05.

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14

12

10

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□ WT

■K O

Figure 10. Fat mass distribution across all regions withinWT and GHSR rats.

4 4

5,7

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- I 5,4 2S J S 5,3

as •“ wo 2 " 52E- T> +• D'^n. 4)DC .2DC .22 ga> c > £ < o &

5,1

5

4,9

4,8

□ WT

■ KO

Figure 11 . Average Total fat (g) mass % normalized to body weight (g) -+SEM.

Experiment 3.1 Metabolic characteristics of the adult WT rats and GHSR KO rats when

exposed to a GTT.

A repeated measures ANOVA on a particular genotype rat model and GTT was

conducted with genotype (WT and GHSR KO) as the between subject factor and AUC time

(AUC 0-15, AUC 15-30, AUC 30-60, AUC 60-120, AUC SUM) as the within subjects factor.

Results revealed a significant main effect for time, F (1.092, 15.283) = 394.917, p < .05, r|2 = .96,

while there was no significant main effect for genotype, F (1, 14) = 1.82, p > .05, rj2 = .12.

Furthermore, there was no significant genotype X time interaction, F (1.092, 15.283) = 1.53, p >

.05, ii2 = .10 (Figure 12-13).

45

1 2 0 0

1000

suVi

+is3Viu3<

800

600

400

200

0

□ WT

■KO

Figure 12. No significant differences found in glucose tolerance test.

12

10

£ ft<u 6</) o u5 400

WT

-KO

15 30min

60 120

Figure 13. AUC curve following bolus of glucose injection in WT and GHSR KO rats.

46

Experiment 4 Arcuate Nucleus orexigenic peptide mRNA differences between WT rats and GHSR KO rats

Genetic mutation o f ghrelin receptor alters basal expression o f hypothalamic neuropeptides AgRP mRNA expression.

Gene expression was examined with qRT-PCR, and results from the assay were

examined with independent group t-tests. These analyses showed that brain tissues within the

ARC in GHSR KO group displayed a significant 6 fold increase in AGRP mRNA expression in

comparison to AgRP mRNA expression (Figure 14) in the hyporthalami o f WT controls t (7)=

1,831 , P < 0.05; see Figure 14). The expression of POMC (Figure 15), leptin receptors and

NPY mRNA, however, was not different between the groups (p. > .05).

AGRP

CDCDCCO

j COtjo

9-8-

7-65H4'

2'

Ho 7

Group

Figure 14. Hypothalamic neuropeptide AgRP mRNA expression in ARC between WT

rats and GHSR KO rats.

47

POMC

6t

Group

Figure 15. Hypothalamic neuropeptide POMC mRNA expression between WT and GHSR KO.

Experiment 5. Hormonal differences between plasma WT rats and GHSR KO rats.

Fasting Active Ghrelin concentration.

GHSR KO displayed trends of reduced active ghrelin within plasma; WT (M= 130,5 SD=136,1)

and GHSR KO (M= 40, 27, SD= 16,35) conditions; t (8)= 1,471, P = 0.0897 (Figure 16).

48

250

ma. 200

a»tm

u<octs'■CV!aU,

150

c 100

□ WT

■KO

p= .08

50

Figure 16. Fasting active plasma ghrelin content between GHSR KO rats and WT rats.

Fasting Glucose concentrations.

No significant differences were found in fasting glucose measures between. WT rats (M

=4.53 SD = 0.37) and GHSR KO rats (M=4.16 SD=0.53) (Figure 17); t (14) = 1.58 p > .05.

4 ,8

> 4 ,6OB

~ 4 ,4OJ (AoU— 4 2

§CO 4QA £« 3 ,8

U .

3 ,6

Figure 17. Fasting glucose concentrations between GHSR KO rats and WT rats.

Fasting insulin concentration.

□ W T

49

Correspondingly, after 3 months of treatment with the high fat diet intake, the fasting

plasma insulin concentration (Figure 18) of GHSR KO rats (M= 2885 SD-393.5) was similar to

WT rats (M= 2491 SD=1505) and) conditions; t (12)= 0,6690 , p > 0.05.

3500

1 3000C/Di 2500 E

2000

| 15005/JCM 1000as

1 500 u.

0

Figure 18. Fasting plasma insulin concentration between GHSR KO and WT.

Fasting leptin concentrations.

Fasting plasma leptin concentrations were similar between WT rats (M= 4974 SD=3147)

and GHSR KO rats (M= 3298, SD= 1569); t (12)= 1,261, p = .11 (Figure 21).

50

7000

| 6000 (Ai 5000

~53C4000

2 ,3000aJwd 2000

£ 1000

□ WT

■KO

0

Figure 19. Fasting plasma leptin concentrations between GHSR KO rats and WT rats.

Fasting triglyceride content.

An altered lipid profile was displayed within GHSR KO rats. GHSR KO rats GHSR KO

(M= 40,27 SD= 16,35 exhibited a reduction in fasting levels of triglycerides relative to WT rats;

WT (M= 156,6 SD= 142); t (8)= 1.486, p < .05 (Figure 20).

250

200■o

E41■o©laatw13d'uH

150

100

50

0

□ WT

■KO

Figure 20. Fasting plasma triglyceride content was decreased in GHSR KO rats relative to WT

rats.

51

Fasting PYY levels.

GHSRK KO rats exhibited no differences in PYY plasma levels relative to WT

littermates; WT (M= 84.73 SD=100.7) and GHSR KO (M= 47.04 SD=25.93) conditions; t (12)=

0.9591, P = 0.1782.

Fasting Growth Hormone concentration.

No significnat differences were observed between GHSRK KO and WT rats’s plasma

GH levels; WT (M= 2.606 SD= 2.342) and GHSR KO (M= 4.255 SD=4.058) conditions; t(12)=

0.9591, p > 0.05.

4 Discussion

Experiment 1: Behavioral characteristic of the FHH rats during Developmental Period.

Effect o f GHSR genetic mutation on food intake in GHSR KO rats.

GHSR KO rats ate the same amount of food as the WT rats while they were developing.

This relationship remained true both in development as well in adulthood. Our results are in

accordance with previous Ghrl/GHSR KO mice models previously reported (Zigman et al., 2005;

Wortley et al., 2004). Thus, this genetic mutation on the ghrelin receptor did not affect food

intake since GHSR KO rats did not eat any less than WT rats.

Effect o f GHSR KO genetic mutation on body weight in GHSR KO rats.

When looking at body weight, our results show GHSR KO rats were smaller from

weaning until the age of 4 months. When matched for aged, GHSR KO rats weighed less

throughout the post weaning periods between 4weeks up until 4 months. As early as the first day

of weaning, they were different by approximately 9 g and that difference persisted throughout

their developmental period and adulthood.

52

A second cohort was examined for regular behavioral measures such as food intake and

body weight following 4 months for an extended 3 weeks period. In this cohort of rats, GHSR

KO rats still gained less weight and consumed less food, when correcting for body weight.

Initial reports o f mice with targeted deletions to the ghrelin gene exhibited no changes in

feeding behavior or body composition when fed standard chow diet, which was somewhat

surprising (Sun et al., 2003; and Wortley et al., 2004). However, additional studies were able to

further analyze the metabolic phenotype of mice lacking ghrelin (Wortley et al., 2005b) or

GHSR (Zigman et al., 2005; Sun et al., 2004) and discovered that both are resistant to diet-

induced obesity when fed a high-fat diet, eating less and preferentially utilizing more stored fat

as an energy substrate than wild-type mice. These data lend support to the notion that ghrelin-

responsive pathways are an important component o f coordinating body-weight control.

Experiment 2-2.1 Behavioral characteristics of the FHH rats during Adulthood Period

including an overnight fast.

Despite no differences in chow food intake between GHSR KO rat and WT rats under

normal conditions, when challenged with an overnight fast, GHSR KO rats ate less than WT rats.

This supports the idea that ghrelin is important for meal initiation and perhaps meal duration. It

is possible that satiety signals have a larger effect on meal duration because ghrelin is not present

in these animals. Future analysis would benefit from investigating other meal factors that might

support this resistance to over eating such as meal rate, and meal duration, or the number and

size of meals in one sitting.

53

Experiment 3. Behvioral characteristics of adult WT and GHSR KO rats when chronically

subjected to HFD.

When fed HFD in adulthood, GHSR KO ate less than WT rats. Previous studies have

demonstrated ghrelin injections, both peripheral and central, increased adiposity (Tschop et al.,

2000; Pierez-Tilve et al., 2011). This is due to ghrelin’s preference for glucose as a metabolic

fuel (Wortley et al., 2004). For instance, Ghrl KO mice exhibit a lower respiratory quotient than

WT mice after only 6 weeks on the HFD diet (Wortley et al., 2004). This suggests that animals

without ghrelin, preferentially use fat as fuel. Perhaps this switch in fuel is coordinated through

stimulating orexigenic neurons and promoting preferential use of glucose as its fuel with ghrelin

intact. When put on a HFD and with a mutation to the ghrelin receptor gene, perhaps a

dysfuntion occurs in the activation of the hypothalmaic pathway in GHSR KO rats or mice

(Briggs et al., 2010). Moreover, another speculation could refer to reports regarding ghrelin’s

stimulatory actions on the reward-VTA pathway to increase the preference and motivation to

obtain palatable food through a Fixed Ratio 1 (FR1) paradigm (King et al., 2011). Nonetheless,

these are speculatory explanations and more studies would be necessary for a better

undesrtanding on why GHSR KO eat less HFD.

The effect o f GHSR KO on body weight and body composition.

Our second cohort of GHSR KO rats gained less weight when fed both standard chow

and HFD. Our results support previous studies measuring the intake o f regular and HFD in

GHSR and ghrelin KO mice (Zigman et al., 2005; Wortley et al., 2004; Wortley et al., 2005; Lin

et al., 2011). GHRS KO rats, however, were not different in amount o f body fat accumulated

than WT rats, even following months of HFD. This similarity between groups could also be due

54

to the background strain in itself being resistant to body weight gain, as well as failing to show a

preference to HFD (Reed et al., 2011). In one study, FHH rats fed a diet similar to the one used

in our study gained less weight than rats from a number o f other rat strains (Reed et al., 2011).

Since they are a normally quite light in comparison to other strains, this may be a confounding

variable. In other words, the background strain may mask the mutation to the ghrelin receptor

gene and prevent us from observing differences between GHSR KO and WT rats.

Experiment 3.1 Metabolic characteristics of the adult WT and GHSR KO rats when

exposed to a GTT.

Interestingly, we found no major differences between GHSR KO and WT rats in the

responses to a glucose tolerance challenge. Previous studies have shown that GHSR KO mice

exhibit lower glucose levels following a glucose challenge suggesting GHSR KO mice display

more efficient glucose clearance and insulin utilization (Sun et al., 2006; Cowley et al., 2005). In

addition, we found no differences in fasting plasma content of glucose and insulin. Perhaps, this

similarity between GHSR KO and WT rats is due to FHH rats consuming large amounts o f HFD

and as a result, fail to become obese. Futhermore, FHH rats may be very efficient at

metabolizing and managing their glucose levels. Future studies would be necessary to gain a

better understanding of their glucose tolerance properties under both conditions o f standard chow

and HFD.

Experiment 4 Arcuate Nucleus orexigenic peptide mRNA differences between WT and

GHSR KO

The effect o f the GHSR mutation within the ARC.

Following prolonged exposure to a high fat diet, GHSR KO rats showed increase mRNA

expression of AgRP in the ARC compared to WT rats also exposed to a high fat diet. Our results

55

are similar to previous data reported by Ligen et al., (2012) noting an increase in AgRP and

POMC within the ARC of GHSR KO rats. We also examined other gene primers including the

leptin receptor (OBRB) and NPY and no significant results were observed. These results have

also been reported by Ligen Li et al., (2012). This could be due to compensatory mechanisms,

which are explained in detail further along this discussion. Thus, our rat model mimics what

occurs in ARC of GHSR KO mice, confirming our rat model is valid to the GHSR KO

phenotype in mice previously characterized.

Experiment 5. Hormonal differences between plasma WT and GHSR KO.

The effect o f GHSR mutation on fasting plasma active ghrelin.

GHSR KO rats demonstrated ghrelin concentrations that tended to be lower than WT rats.

This could be because ghrelin levels are reduced in animals exposed to a high fat diet (Perreault

et al., 2003). It is possible that GHSR KO rats are slightly more affected by this than WT rats. A

former study observed that both ghrelin secretion and the sensitivity to the orexigenic effects of

exogenous ghrelin were reduced in obese mice when compared to lean mice fed standard chow

(Briggs et al., 2010). Conversely, the sensitivity to ghrelin is reduced in obesity but improves

after weight loss (Perreault et al., 2004). Therefore, our findings coincide with previous studies

demonstrating that HFD decreases ghrelin levels in rodents and that plasma ghrelin levels also

are lower in obese humans (Handjieva-Darlenska & Boyadjieva 2009; Cummings et al., 2001).

Effect o f GHSR KO on fasting plasma insulin content.

It is well known that obesity or in particular, a chronic HFD, often leads to insulin

resistance, which can further lead to pancreatic b-cell failure and type 2 diabetes (Kahn et al.,

2006). Past studies have reported the deletion of ghrelin increases insulin secretion in response to

glucose challenge and subsequently increases peripheral insulin sensitivity (Sun et al., 2006).

56

Some studies have shown that there is insulin resistance shown within WT mice but not in

GHSR KO mice. One study from Sun et al., (2011) examined rats at different age periods. WT

rats (4 months old) became severely insulin resistant in response to a bolus o f insulin, while old

GHSR KO mice (18, 20, 24 and 26 months old) were significantly more responsive to an insulin

challenge when compared to age-matched WT mice. Moreover, GHSR KO rats maintained a

response curve similar to that of insulin-sensitive young mice (Ligen Li et al., 2011). Together

these functional tests suggest that GHSR KO mice have improved peripheral insulin sensitivity

and this GHSR KO effect has been shown to improve as a function of aging (Sun et al., 2007). It

would be interesting if future studies could investigate Insulin Tolerance Tests in these FHH

GHSR KO and WT rats. This way, a more complete picture could be uncovered regarding

ghrelin’s role within glucose homeostasis o f this background strain. Lastly, a comparison

between strains with these measures would aid in better understanding the strain effect.

The effect o f GHSR KO on fasting plasma leptin profile.

Leptin is one of several hormones secreted by adipose tissue and is often correlated with

the amount of lipids stored in WAT. Our results reveal no significant differences in plasma leptin

content between GHSR KO and WT rats. This may be not surprising given that GHSR KO and

WT rats had similar body fat content after a 15-week exposure to the HFD. Moreover, our results

are also consistent with previous literature stating leptin level values were unchanged in GHSR

KO mice following 19 weeks of standard chow and 3 weeks of HFD exhibited (Zigman et al.,

2005).

Lipid profile

White adipose tissue stores energy in the form of triglycerides and supplies energy to the body as

ATP through lipolysis/B oxidation (Amati et al., 2009; Khan et al., 2006). To determine whether

57

GHSR KO rats maintained a healthier lipid profile during the chronic HFD exposure, the fasting

plasma lipid profiles of WT and GHSR KO rats were analyzed. Several levels o f the hormones

secreted by adipose tissue often correlate with the amount o f lipids stored in WAT; specifically

visceral adiposity (Amati et al., 2009). Despite no significant differences in fat mass distribution,

an altered lipid profile was manifested in GHSR KO rats. In our study, WT rats displayed more

plasma triglyceride content than GHSR KO rats after exposure to the HFD. Therefore, our results

support the idea that deletion of GHSR improves lipid profiles.

The effect o f GHSR gene mutation on fasting plasma peptide YY profile.

Our results reveal GHSR KO rats do not show any differences in satiety hormones

relative to WT littermates. These findings are consistent with the other satiety hormone such as

leptin. Therefore, we cannot attribute their food consumption to differences in satiety hormones.

Other studies have reported PYY is reduced in GHSR KO mice when subjected to HFD. PYY’s

role in energy homeostasis mainly occurs when an energy restriction exist. While ghrelin

increases in these negative energy balances, PYY decreases in the circulation and the reverse

occurs in cases o f positive energy balance (Stanley et al., 2005). This however, was not the case

in the current study and both WT and GHSR KO rats had similar plasma concentrations o f leptin

and PYY.

The effect o f GHSR gene mutation on fasting plasma growth hormone.

Our studies show no effects in GH concentrations between GHSR KO and WT rats.

Presumably, we would have expected a difference in growth hormone given that ghrelin

increases the release of GH. However, GH is not only secreted in response to ghrelin but other

GH secreting factors as well including growth hormone releasing hormone (GHRH). Therefore,

58

GHSR KO rats may have normal concentrations o f GH because other factors may maintain GH

concentrations normal.

4.6 Compensatory Mechanisms

It has been speculated for quite some time that compensatory mechanisms may develop

from birth when organisms are bom with a gene mutation early in life. This could serve as and

adaptation for survival because this loss of function is a global mutation found in every tissue.

Therefore the lack of effects in our study is assumed to be attributed to a compensatory

mechanism(s). The only ways to discover if this is the case would be to eliminate this confound

and induce the KO of the ghrelin receptor gene in adulthood. Possibilities to delete genes

encoding for the ghrelin receptor during adulthood include more advanced conditional gene

knockouts such in-vivo Cre-Lox recombination. Cre-Lox recombination is commonly used to

carry out deletions for tissue-specific knockouts and also time-specific knockouts (Guo,

Deshmukh & Van Duyne, 1997). This is not possible in conventional gene knockouts since the

gene is knocked out in early embryonic cells. Briefly, cre-recombinase is an enzyme that

recombines in a particular LoxP sequence the genomic DNA in order for and endogenous gene

such as the ghrelin recetpor gene to be deleted (Guo et al., 1997; Zigman et al., 2005).

Viral Vectors can also manipulate DNA in adulthood. They are used to deliver genetic

material into cells (Robbins & Ghivizzani, 1998). That is how protein-coding genes are

expressed, such as marker genes like Green Fluorescent Protein in order to label and track cells

of interest. All viruses will attack their hosts and introduce their genetic material into the host

cell as part of their replication cycle (Robbins et al., 1998). This genetic material contains basic

instructions of how to reproduce more copies of these viruses. Once these are carried out, more

cells become infected (Robbins et al., 1998).

5 9

Recently an anti-ghrelin vaccine carrying a viral vector was designed to trigger the

production of antibodies to neutralize ghrelin and was administered to DIO and wildtypes mice

(Andrade, Couselo Carreira, Ribeiro, Texeria et al., 2011). The goal was to prevent ghrelin from

communicating with the brain. This would help in maintaining weight loss since circulating

ghrelin levels rise following weight loss.

Together, along with ghrelin antagonist studies, these in vivo techniques could overcome

the confound of compensatory mechanism and be able to bypass this background strain effect in

order to detect the gene mutation effect on the phenotype.

Resistance to diet-induced obesity.

All together, data shows rat with GHSR mutation eat less on a HFD than WT littermates

and as a result, weight less. Possibly, this greater response is due to a meal preparatory factor in

which ghrelin signals and prepares the body for the fuel it’s about to consume. Emerging

evidence reports ghrelin’s ability to interpret energy reserve amounts and in response, build fat

reserves in times of need (Wortley et al., 2005). Without this GHSR signaling pathway, the

process is compromised and animals are less able to interpret and signal when energy stores are

low. Not having GHSR receptors has potential beneficial effects on preventing the reward­

pathway to become overly stimulated and override homeostastic signals. This is the case when

high calorically dense food is easily accessible in large quantities such as our western

environment today. Therefore, in this context, ghrelin receptor antagonists could prove useful in

controlling adiposity in human obesity associated with a HFD.

Despite male GHSR KO rats displaying comparable body compositions to those of their

wild-type littermates following 3 months o f exposure to a HFD, this condition resulted in

significantly less daily accumulation of both body weight and triglyceride content in GHSR KO

6 0

rats as compared with littermate controls. Our future studies would benefit from untangling

where this weight loss and lower triglyceride content could be attributed. Comparing the

percentage of fat in this particular background strain when fed a HFD to the percentage o f lean

body mass including bones and muscles and other fat free tissues such as organs could provide a

better idea to pursue other mechanism which underlie their body weight loss. Other speculations

could include increased locomotor activity or increased sympathetic tone due to their

hypertensive nature.

4.7 Strengths

The significance of our study is that it can provide a valid GHSR KO rat model that

displays a similar phenotype as that seen in mice with similar mutations. To our knowledge,

there is only one other report using the GHSR KO rat model examining ghrelin’s role in drug

addiction behaviors (Clifford, Rodrigues, Schul, Hughes, Kniffin et al., 2011). However, the

characterization of this rat model is yet to be determined. Therefore, this thesis presents data that

validates this particular rat model. Future studies may ensue with more background strain

phenotype available.

4.8 Limitations

The background strain used to generate the FHH rat, as shown by our studies, is resistant

to gaining weight and does not overeat when given a high fat diet. This may pose a major

limitation to this rat model, and it would be advisable to cross breed this strain onto a

background that is more prone to obesity like the Long Evans Rat. Nevertheless, and in spite of

this limitation, our studies show clear metabolic and behavioral differences between the GHSR

KO and WT FHH rats that match those seen in GHSR mutant mice.

61

4.9 Validation of DIO Rat Model

In summary, our GHSR FHH KO rat models many of the features o f the obesity

syndrome and our results are similar to previous work (Zigman et al., 2005; Wortley et al., 2004,

Lin et al., 2011). The mechanisms by which ghrelin promotes food intake are multifaceted and

include to a great extent, enhancing the rewarding properties of certain foods (Abizaid et al.,

2009; Chuang et al., 2011). Therefore, targeting the gene encoding for ghrelin would presumably

affect feeding behaviors. As it would be expected, GHSR KO ate less food and gained less body

weight under HFD conditions. As a result, GHSR KO displayed a significant decrease in

triglyceride content as well as reduction trends in ghrelin suggesting a dysregulation in theis

appetite-regulatory hormone. Moreover, GHSR KO rats displayed an increase in AgRP mRNA

expression. This finding also supports the notion of a compensatory mechanism.

Overall, this model would become a pivotal model for understanding the interplay

between genetic background and environmental challenges such as high-fat/high-calorie diets

that predispose to the development o f metabolic syndrome (Collins et al., 2004). Also, this

animal model could be used to expand diet conditions and exposure animals to other diets such

as high fructose or high protein in order to depict and untangle more of the diets influence on the

overall energy balance of the FHH GHSR KO rat.

5 Conclusions

We conclude to have demonstrated that the genetic disruption of the gene encoding for

ghrelin receptors system with FHH rats leads to a similar phenotype found within GHSR KO

mice studies. In particular, a series of mice studies show that the deletion of the ghrelin/GHSR

gene reduces food intake, body weight and adiposity, through reduction of appetite and

62

augmentation of energy expenditure and fat catabolism (Wortley et al., 2004; Zigman et al.,

2005).

Our results indicate that when accounting for body weight differences, GHSR KO rats

consume significantly less food than their WT littermates. This was observed regardless o f the

diet they consumed. Furthermore, when challenged with an overnight fast (to observe rebound

feeding responses), GHSR KO rats consumed significantly less than WT suggesting ghrelin’s

short-term effects o f initiating meals and regulating food intake remained intact. Within the

GHSR KO hormonal profiles, our results support previous findings demonstrating reduced

triglyceride content relative to WT, which would presumably reflect their lower levels o f white

adipose tissue and lower body weights. Furthermore, ARC mRNA expression reveals a

dysregulation in orexigenic neuropeptides stimulated by ghrelin.

Based on lower body weight accumulation exhibited in both GHSR KO and WT rats

(after HFD), our data suggests the possibility that the resistance to diet-induced obesity may be

further influenced by the FHH background strain. As a result, this background strain may

potentially mask differences produced by the GHSR gene mutation. However more

characterization is needed in order to confirm this speculation. Possibly even a comparison

analysis with other strains previously established to be more prone to obesity such as the Long

Evans or Wistar rat would be beneficial.

Together, our data suggests that endogenous ghrelin is essential for the maintenance of

normal levels or patterns o f food intake, or increases in food intake after a fast. Thus, the absence

of ghrelin receptors, due to genetic deletion leads to the inability for ghrelin to bind to their

receptors and as a result, exert its functions. Thus, our data validates utilization of the GHSR KO

rat as a model to target diet-induced obesity since rats deficient o f ghrelin’s receptor seem to

63

exhibit a protective phenotype resisting high fat diet induced-obesity. This type of research is

important and relevant because of the high prevalence and associated metabolic diseases when

ghrelin-signaling pathway is dysfunctional including obesity and Type 2 Diabetes. For

individuals at highest risk of the complications o f severe obesity, such findings provide a starting

point for attaining more rational mechanism-based therapies. Clinical implications could prove

useful in controlling adiposity through ghrelin receptor antagonists in human obesity associated

with HFD.

64

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Appendix A.

Different phenotypes o f inbred mouse strains with diet or genetically induced obesity. Strain Characteristics Crossed with obese mice, and so forth

" " T " TLep ° ° mice exhibit obesity but not diabetes Lep oh ob mice exhibit obesity, severe diabetes

Lep ohob mice exhibit obesity, severe diabetes

Homozygous fo r db allele db mice display mild hyperglycemia with marked hyperinsulinemia and develop hypoglycemia leading to sudden death Lepobob mice exhibit obesity with severe diabetes

Not reported

Lep oh oh mice exhibit reduced adiposity and increased thermogenesis and are fertile.Not reported

Not reported

Not reported Not reported

Not reported

C57BL/6J HFD-induced obesity and diabetes C5 7BLKS/J HFD-induced obesity and diabetes

Weaker than C57BL/6J DBA/2 More glucose tolerance than

C57BLK/6 on a HFD 129sv Low insulin; more glucose tolerance

than other strains on a HFD

BTBR Abdominal obesity with peripheralbut not hepatic insulin-resistance.A/J Low glucose level even on a HFD;

Obesity and diabetes-resistant BALB/c Similar to A/J; glucose tolerance

C3H High glucose tolerance with robustInsulin secretion.

AKR Sensitive to DIO with hyperinsulinemiaand insulin resistance.

CAST/ei Lean at 12 weeks on HFDNonobese 45% fa t diet-induced transient hyper- Diabetic glycemia with severe obesity.New Resembles metabolic syndrome in Zealand humans; HFD DIO and hyperglycemia ObeseFVB High glucose levels with lower levels o f

insulin on normal chow

Kuo Obesity: hyperleptinemia; increase Kondo glucose and hbAlc; hypersinsulinemia

Similar to C57BLKS/JLep oh ob mice TallyHo Natural model o f obesity with type 2

diabetesNagoya Exhibit obesity; all males and 1/3

o f females exhibit glucose intolerance

Lep db ob mice display insulin resistance, hyperglycemia and severe hyperinsulinemia compared to C56BL/6 Ay mutation in agouti results in diabetes nephropathy

Not reported

Not reported

Modified from Keizo Kanasaki and Daisuke Koya, (2011). Biology o f Obesity: Lessons from Animal

Models o f Obesity.

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Appendix B.

Tecklad Global 14% Protein Rodent Maintenance Diet.

Macronutrients Percentage Amount

Crude Protein % 14.3

Fat (either extract) % 4.0

Carbohydrate % 48.0

Crude Fiber % 4.1

Neutral Detergent Fiber % 18.0

Ash % 4.7

Energy Density kcal/g (KJ/g) 3.4(12.9)

Calories from Protein % 20

Calories from Fat % 13

Calories from Carbohydrate % 67

81

Appendix C.

Open Source 60% High-Fat Diet.

Macronutrients gm% kcal%

Protein 26.2 20

Fat (either extract) 34.9 60

Carbohydrate 26.3 20

Total

Kcal/gm 5.2

82

Appendix D.

Gene Primer SequenceNPY

Sense 5 '-TCCGCTCTGCGAC ACTAC AT-3'Antisense 5 '-GGAAGGGTCTT C AAGCCTT GT-3'

AgRPSense 5' -GCT CC ACT G AAGGGC AT C A-3'Antisense 5 '-TAGC ACCTCCGCC AAAGCT-3'

POMCSense 5 -GCTCAAGGTCCTTCCTGGTG-3'Antisense 5 -GCCCTGGATTGAATCACGCC-3'

OB-RbSense 5'-GCTGGAAGCCTGTCGTACTCTTCAC-3'Antisense 5 '-TAC ACTGCGTC ATAGGTA AACTTCCCTC-3'

Beta-ActinSense 5 '-GTGCC ACC AGAC AGC ACTGTGTTG-3'Antisense 5' -T GG AG A AG AGCT AT G AGCT GCCT G-3'

GAPDHSense 5'-AGCAATGCCTCCTGTACCAC-3'Antisense 5'-AAGC AGGG AT GAT GTTCTGG-3'

qRT-PCR primers: NPY, neuropeptide Y; AgRp, agouti-related protein; POMC, pro- opriomelanocortin; LepR, leptin receptor; Control Primers: Beta-Actin; GAPDH.

83